CN104903349A

CN104903349A - Il-6 antagonists and uses thereof

Info

Publication number: CN104903349A
Application number: CN201380058297.0A
Authority: CN
Inventors: M.M.施密特; D.V.厄布; T.M.巴恩斯; E.S.弗法恩; A.蒂斯代尔
Original assignee: Eleven Biotherapeutics Inc
Current assignee: F Hoffmann La Roche AG; Carisma Therapeutics Inc
Priority date: 2012-11-08
Filing date: 2013-11-08
Publication date: 2015-09-09
Anticipated expiration: 2033-11-08
Also published as: KR102149206B1; HK1211599A1; IL238238A0; EP2917238A1; US20150125468A1; RU2670943C2; US20180222975A1; JP6480338B2; WO2014074905A1; KR20150079789A; CN104903349B; US9951130B2; US11459386B2; AU2013342163A1; SG11201502876RA; AU2013342163B2; RU2015121755A; CA2886987C; MX2015005831A; CA2886987A1

Abstract

IL-6 antagonists are provided that are specific for binding to site II of IL-6. Methods of using such inhibitors to treat IL-6 related diseases, e.g., disease of the eye such as diabetic macular edema are disclosed.

Description

IL-6 antagonist and application thereof

Related application

This application claims the right of priority in the U.S. Application Serial Number 61/723,972 of submission on November 8th, 2012 and the U.S. Application Serial Number 61/831,699 in submission on June 6th, 2013.The full content of aforementioned each application is incorporated to the present invention by reference.

Invention field

Field of the present invention relates to IL-6.More specifically, described field relates to the conditioning agent of IL-6 and is treating as the application in illness in eye.

Background technology

IL-6 is a kind of pleiotropic cytokines in inflammation, hematopoiesis (hematopoiesis), vasculogenesis, cytodifferentiation and neuronal survival with report effect.

Summary of the invention

The present invention relates to the IL-6 antibody and fragment with some feature (comprising the site II specific binding to IL-6), and the derivative of this antibody-like and fragment, and use the method for this antibody, fragment and derivative.Therefore, the present invention relates to the antibody of the site II of specific binding IL-6, fragment or derivatives thereof.In some embodiments, antibody, fragment or derivative can with the K of 240pM or lower _din conjunction with IL-6.In some embodiments, antibody, fragment or derivative have 70 DEG C or higher T _m.In some embodiments, antibody, fragment or derivative can with the K of 240pM or lower _din conjunction with IL-6, and there is 70 DEG C or higher T _m.In some cases, antibody or fragment or derivatives thereof are in conjunction with at least one of R24, K27, Y31, D34, S118 or V121 of people IL-6; In some cases, antibody or fragment or derivatives thereof can in conjunction with R24, K27, Y31, D34, S118 and V121 of people IL-6.In embodiments, antibody or fragment or derivatives thereof can in conjunction with at least 1 in R24, K27, Y31, D34, S118 and V121 of people IL-6, at least 2, at least 3, at least 4 or at least 5.

One aspect of the present invention provides can the antibody of site II of specific binding people IL-6 or its fragment (as its Fab).

In embodiment, antibody or its fragment (as its Fab) can with the K of 200pM or lower _din conjunction with IL-6.

In embodiment, antibody or its fragment (as its Fab) can with the K of 200pM or lower _din conjunction with IL-6 and/or have 70 DEG C or higher T _m.

In embodiment, antibody or its fragment (as its Fab) can with the K of 200pM or lower _din conjunction with IL-6 and/or have 80 DEG C or higher T _m.

In embodiment, antibody or its fragment (as its Fab) are in conjunction with at least one in R24, K27, Y31, D34, S118 and V121 of people IL-6.In embodiment, antibody or its fragment (as its Fab) are in conjunction with at least two in R24, K27, Y31, D34, S118 and V121 of people IL-6.In embodiment, antibody or its fragment (as its Fab) are in conjunction with at least three in R24, K27, Y31, D34, S118 and V121 of people IL-6.In embodiment, antibody or its fragment (as its Fab) are in conjunction with at least four in R24, K27, Y31, D34, S118 and V121 of people IL-6.In embodiment, antibody or its fragment (as its Fab) are in conjunction with at least five in R24, K27, Y31, D34, S118 and V121 of people IL-6.In embodiment, antibody or its fragment (as its Fab) are in conjunction with R24, K27, Y31, D34, S118 and V121 of people IL-6.

The invention provides a kind of antibody on the one hand (as the antibody be separated, monoclonal antibody as being separated) or its fragment (as its Fab), it comprises (a) VH CDR1 as shown in SEQ ID NO:4, VH CDR2 as shown in SEQ ID NO:5, and the VH CDR3 as shown in SEQ ID NO:6; And optionally (b) VL CDR1 as shown in SEQ ID NO:7, VL CDR2 as shown in SEQ ID NO:8, with the VL CDR3 such as shown in SEQ ID NO:9, wherein said antibody or its fragment (as its Fab) can specific binding people IL-6.In embodiments, antibody or its fragment comprise identical with the CDR sequence shown in SEQ ID NO:4, SEQ ID NO:5 with SEQ ID NO:6 respectively, or are altogether no more than 1,2,3,4 or 5 amino acid whose heavy chain CDRs VH CDR 1, VH CDR2 and VH CDR3 with the difference of described CDR sequence.In embodiments, antibody or its fragment comprise identical with the CDR sequence shown in SEQ ID NO:7, SEQ ID NO:8 with SEQ ID NO:9 respectively, or are altogether no more than 1,2,3,4 or 5 amino acid whose light chain CDRs VL CDR 1, VL CDR2 and VL CDR3 with the difference of described CDR sequence.

The invention provides a kind of IL-6 antibody (the IL-6 antibody as being separated) or its fragment (as its Fab) on the one hand, it can with the K of 240pM or lower _d(as determined by surperficial plasmon resonance) is dissociated with people IL-6.In embodiments, antibody can with in the IC50 of 255pM or lower and IL-6 active, as by external HEK-Blue ^tMiL-6 assay method measures.

The invention provides a kind of IL-6 antibody (the IL-6 antibody as being separated) or its fragment (as Fab) on the one hand, it can comprise antibody or its fragment (as Fab) of SEQ ID NO:1 and SEQ ID NO:2 or comprises the antibody of SEQ ID NO:3 and SEQ ID NO:2 or the combination of its fragment (as Fab) and people IL-6 by competitive inhibition.

In embodiments, antibody or its fragment (as its Fab) have the unit price Kd of 2nM or higher under pH5.5.

In embodiments, antibody or its fragment (as its Fab) are IgG2 antibody.

In embodiments, the FcRn that antibody or its fragment (as its Fab) have a change compared with reference antibody (reference antibody) combines.In embodiments, compared with reference antibody, it is decline that described FcRn combines.In embodiments, the Fc structural domain of antibody or fragment changes compared with reference antibody.

The invention provides a kind of IL-6 antibody on the one hand (can in conjunction with IL-6, antibody as the site II of people IL-6) or its fragment, it has the Fc structural domain of modification and the FcRn showing reduction compared with the corresponding antibody with wild-type Fc structural domain combines.

In embodiments, antibody or its fragment (as its Fab) are included in the one or more sudden change in H311, I254 and H436 of SEQ ID NO:23.

In embodiments, antibody or its fragment (as its Fab) are included in the two or more sudden change in H311, D313, I254 and H436 of SEQ ID NO:23.

In embodiments, antibody or its fragment (as its Fab) are included in three or more the sudden change in H311, D313, I254 and H436 of SEQ ID NO:23.

In embodiments, antibody or its fragment (as its Fab) are included in each sudden change in H311, D313, I254 and H436 of SEQ ID NO:23.

In embodiments, antibody or its fragment (as its Fab) comprise the one or more sudden changes (as 1,2,3 or 4 sudden change) being selected from the group be made up of H311A, H311E, H311N, D313T, I254A, I254R and H435A.

In embodiments, antibody or its fragment (as its Fab) are separated.

In embodiments, antibody is monoclonal antibody or its fragment (as its Fab).Antibody is human monoclonal antibodies in embodiments.

In embodiments, antibody is the monoclonal antibody or its fragment (as its Fab) that are separated.

In embodiments, antibody (monoclonal antibody as being separated) comprises SEQ ID NO:23.

Present invention also offers a kind of antibody or its fragment (as Fab) (the IL-6 antibody described in the present invention or its fragment), or comprise the composition of this antibody or its fragment, be used for the treatment of the disease (suffering from the experimenter of IL-6 relative disease as being used for the treatment of, as people experimenter) that IL-6 is relevant.In embodiments, described disease is the eye disease that the IL-6 level to raise in vitreum is feature.In embodiments, described disease is diabetic macular edema (diabetic macular edema, DME), diabetic retinopathy (diabetic retinopathy), uveitis (uveitis), xeropthalmus (dry eye disease), uveitis (uveitis), age-related macular degeneration (age-related macular degeneration, AMD), proliferative diabetic retinopathy PDR (proliferative diabetic retinopathy, PDR), retinal vein occlusion (retinal vein occlusion, RVO), optic neuromyelitis (neuromyelitis optica, NMO), corneal graft (corneal transplant), corneal abrasion (corneal abrasion) or eyes physical damnification (physical injury to the eye).In embodiments, described disease is DME.In embodiments, described disease is xeropthalmus.In embodiments, described disease is dry eye syndrome.In embodiments, described disease is uveitis.In embodiments, described disease is AMD.In embodiments, described disease is PDR.In embodiments, described disease is PDR.In embodiments, described disease is corneal transplantation, corneal abrasion or eyes physical damnification.In embodiments, antibody or its fragment (as Fab) are suitable for the vitreum being delivered to eyes.In embodiments, antibody or its fragment (as Fab) are delivered to the vitreum of eyes.

Present invention also offers a kind of method for the treatment of IL-6 relative disease, described method comprises uses IL-6 antibody or its fragment (as its Fab) to experimenter, IL-6 antibody such as described in the invention or its fragment.In embodiments, IL-6 antibody or its fragment (as its Fab) is used to treat significant quantity.In embodiments, IL-6 relative disease is the eye disease that the IL-6 level to raise in vitreum is feature.In embodiments, described disease is diabetic macular edema (DME), diabetic retinopathy, uveitis, dry eye syndrome, xeropthalmus, uveitis, age-related macular degeneration (AMD), proliferative diabetic retinopathy PDR (PDR), retinal vein occlusion (RVO), optic neuromyelitis (NMO), corneal graft, corneal abrasion or eyes physical damnification.In embodiments, antibody or its fragment (as its Fab) are suitable for the vitreum being delivered to eyes.In embodiments, antibody or its fragment (as its Fab) are delivered to the vitreum of subject eye.In embodiments, IL-6 relative disease is diabetic macular edema and antibody or its fragment is delivered to the vitreum of subject eye.

The invention provides the carrier that one comprises the sequence of coding IL-6 antibody of the present invention or its fragment (as its Fab) on the other hand.In embodiments, described carrier comprises the sequence of coding SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

Another aspect of the present invention provides a kind of cell of expressing IL-6 antibody of the present invention or its fragment (as its Fab) sequence.In embodiments, one or more in described cell expressing SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

In yet another aspect, the present invention relates to the method reducing the systematic effect suppressing IL-6 in experimenter, described method comprises uses the activity that can suppress IL-6 to experimenter, and has antibody or its fragment of the Fc activity of reduction compared with the corresponding antibody of wild-type Fc structural domain or its fragment.In some cases, reduce in described experimenter and suppress the method for systematic effect of IL-6 to comprise to use such as at a low ph (as pH5.5) to experimenter that there is the IL-6 antagonist combined higher than the FcRn of 1 μM.

As used in the present invention, term " antibody " is identical with immunoglobulin (Ig) implication, and is known in the art.Term antibody is not by the restriction of any ad hoc approach of Dispersal risk.Such as, term antibody comprises, particularly recombinant antibodies, monoclonal antibody and polyclonal antibody.As used in the present invention, antibody is a kind of tetramer, unless and in addition openly, each antibody is by two to identical polypeptide chain composition, and wherein often pair has a light chain and a heavy chain.The aminoterminal of each chain is included in about 100 to 120 or more the amino acid whose variable regions played a major role in antigen recognition.The carboxyl terminal of each chain is included in the constant region played a major role in antibody mediated effect subfunction (antibody effector function).The kind of people's light chain is called as kappa and lambda light chain.Heavy chain classes is mu, delta, gamma, alpha or epsilon, and defines the isotype of antibody.The isotype of antibody is respectively IgM, IgD, IgG, IgA and IgE.In light chain and heavy chain, variable region and constant region are connected by about 12 or more amino acid whose " J " district, and heavy chain also comprises one about 3 or more individual amino acid whose " D " districts.

Each heavy chain/light chain variable region to (VH and VL) forms antigen binding site respectively.Therefore, such as complete IgG antibody has two basic change site.Except difunctional or bi-specific antibody, described two basic change site is identical.

Heavy chain of antibody and the variable region of light chain show the same general structure of the relative conserved framework regions (FR) be connected by 3 hypervariable regions (also referred to as complementary determining region or CDR).Term " variable " refers to that between antibody, some site of variable domain exists difference widely in sequence, and these sites relate to combination and the specificity of each specific antibodies and its specific antigen.Mutability (variability) is mainly arranged in CDRs, and it by the framework region (FRs) of high conservative more separately.According to Kabat Sequences of Proteins of Immnunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) or Chothia and Lesk, J Mol Biol 196:901-917 (1987); The amino acid whose distribution of each structural domain is carried out in the definition of Chothia etc., Nature 342:878-883 (1989) (which describing method well known in the art).

" wild-type " can refer to allelotrope the most general or kind in a colony, or refer to the antibody of the animal come from without operation, the latter be with the allelotrope of the operation from certain form or polymorphism or variant or derivative in contrast, described operation is such as mutagenesis, uses recombination method etc. to change the amino acid of this antigen binding molecules.

Term " antibody fragment " refers to complete or total length chain or antibody a part, is generally target land or variable region.The example of antibody fragment includes but not limited to Fab, Fab ', F (ab ') 2 and Fv fragment." function fragment " or " analogue of the site II antibody of anti-IL-6 " be can prevent or essence reduce IL-6 be attached to acceptor ability, reduce IL-6/IL-6R mixture and be attached to the ability of gp130 or reduce the ability of ligand binding to gp130 or start signal (initial signaling).As used in the present invention, function fragment is usually identical with " antibody fragment " implication, and can refer to prevent with regard to antibody or essence reduce IL-6 be attached to acceptor ability, reduce IL-6/IL-6R mixture and be attached to the ability of gp130 or reduce the fragment of ligand binding to gp130 or start signal ability, such as Fv, Fab, F (ab ') 2 etc.

" derivative " of antibody be can specifically in conjunction with IL-6 site II's and share sequence with the site II antibody of IL-6, such as sharing can specifically in conjunction with the polypeptide of at least one CDR of the antibody of the site II of people IL-6.

" competition " refers to that first antibody or its fragment can be combined with second antibody or its fragment competes, when making not exist with second antibody first antibody combination compared with, second antibody exist under, the reduction that first antibody is combined with its epi-position can be detected.In some cases, this term also refers to that the combination of second antibody and its epi-position is detectable reduction under first antibody exists.In nonrestrictive example, the mechanism of this competition is by steric hindrance, conformational change or be combined with common epitope.

Term " Percentage of sequence identity " in nucleotide sequence content refer to when comparison with obtain maximum to two sequences during correspondence in identical residue.The length that sequence iden compares can exceed at least about 9 Nucleotide, such as, at least about 18, at least about 24, at least about 28, at least about 32, at least about 36 or at least about 48 or more Nucleotide.The identity of algorithm measurement nucleotide sequence as known in the art can be used.Such as, the many nucleotide sequences of FASTA, Gap or Bestfit (Wisconsin Package 10.0 version, Genetics Computer Group (GCG), Madison, WI) can be used.FASTA, comprises such as program FASTA2 and FASTA3, provides comparison and Percentage of sequence identity (Pearson, the Methods Enzymol 183:63-98 (1990) of best overlapping region between inquiry and search sequence; Pearson, Methods Mol Biol 132:185-219 (2000); Pearson, Methods Enzymol 266:227-258 (1996); Pearson, J Mol Biol276:71-84 (1998); Be incorporated to by reference herein).The default parameter of usual use specific program or algorithm.Such as, Percentage of sequence identity between FASTA and default parameter thereof (word length be 6 and NOPAM coefficient for score matrix) definite kernel acid sequence can be used, or use GCG the 6.1st edition Gap with default parameters provided to determine, be incorporated to by reference herein.

Term " Percentage of sequence identity " in aminoacid sequence refers to when comparison is to obtain maximum correspondence, residue identical in two sequences.The length of sequence iden comparison can exceed at least 5 amino-acid residues, such as, at least 20 amino-acid residues, at least 30 amino-acid residues, at least 50 amino-acid residues, at least 100 amino-acid residues, at least 150 amino-acid residues or at least 200 an or more amino-acid residue.Usual use sequence analysis software measures the sequence iden of polypeptide.For determining that the algorithm of Percentage of sequence identity is known.Such as can use FASTA, Gap or Bestfit (Wisconsin Package the 10.0th edition, Genetics Computer Group (GCG), Madison, WI) comparing amino acid sequence.The measurement of other similaritys of modifying that Protein analysis software uses comparison multiple replacement, lack and comprise conservative amino acid displacement carrys out matching sequence.Such as, GCG contains such as " Gap " and " Bestfit " program, and they can with deciding closely-related polypeptide (such as from the homologous polypeptide of the biology of different plant species) or the sequence homology between wild-type protein and its analogue or sequence iden together with default parameters.See, such as GCG the 6.1st edition (University of Wisconsin, Madison, WI).Peptide sequence also can use the FASTA (see GCG the 6.1st edition) of acquiescence or proposed parameter to carry out comparison.FASTA (such as FASTA2 and FASTA3) provides comparison and Percentage of sequence identity (Pearson, the Methods EnzymoI183:63-98 (1990) of best overlapping region between inquiry and search sequence; Pearson, Methods Mol Bioi 132:185-219 (2000)).When will by sequence from during containing a large amount of database comparison from the sequence of different biology, operable another kind of algorithm be BLAST, such as blastp or tblastn, the default parameters that service routine provides.See such as Altschul etc., J Mol Bioi 215:403-410 (1990); Altschul et al., Nucleic Acids Res 25:3389-402 (1997).

When sample demonstrate the polypeptide of single kind at least about 60 or 75% time, albumen or polypeptide are " substantially pure, " " basic homogeneous, " or " basic purifying ".Polypeptide or albumen can be monomer or polymer.Substantially pure polypeptide or albumen can comprise about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% sterling; Such as, a substantially pure polypeptide or albumen be 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% pure.The purity of albumen or uniformity are evaluated by any appropriate means, as the polyacrylamide gel electrophoresis of protein sample, then pass through decompression capillary electrophoresis, peptide mapping or the glycan collection of illustrative plates in observation one or more band (as on the gel dyeed) relevant to albumen or polypeptide, size exclusion HPLC, cationic exchange HPLC, SDS.Currently known methods of the prior art or other way of purification such as can be used to realize high resolving power.

When referring to nucleic acid or its fragment, term " basic simlarity " refers to when inserting or disappearance with suitable Nucleotide, when carrying out best comparison with another nucleic acid (or its complementary strand), by any known sequence iden algorithm, the identity of nucleotide sequence of the nucleotide base measured as FASTA, BLAST or Gap is at least about 85%, at least about 90%, and at least about 95%, 96%, 97%, 98% or 99% sequence iden.

When being used on polypeptide, term " basic identity " or " basic simlarity " refer to when two peptide sequences are with when such as GAP or BESTFIT program uses default gap weight to carry out best comparison, share the sequence iden at least about 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99%; Such as, the sequence iden of 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99%.In some embodiments, the difference of not identical resi-dues is that conserved amino acid changes.

" treatment significant quantity " refers to that the therapeutical agent of using this dosage will alleviate at least one sign of institute's disease therapy or symptom or increase or raising and can be used for prevention and or the result for the treatment of of another therapy (as another kind of therapeutical agent) for the treatment of IL-6 relative disease.Be to be understood that treatment significant quantity can be used or as long-term treatment by multiple doses within the limited time.

" treatment " refers to the method alleviating sign or symptom or disease.

As used in the present invention, term " disease " comprises disease and illness.

Each patent document that the present invention mentions and the whole of Science article disclose, and at this these patent documents quoted and Science article, are incorporated herein clearly by reference for all objects.

Other characteristics and advantages of the present invention is discussed in more detail below.

Embodiment

Following non-limiting examples further illustrates embodiments of the present invention of the present invention.

Embodiment 1: the blocking-up of checking local IL-6 in choroidal neovascularization (CNV) model

Such as, in order to determine local I L-6 blocks whether effectively can treat disease of eye, and diabetic macular edema (DME) or moist AMD, anti-IL-6 antibodies is applied topically in choroidal neovascularization model system.The CNV model (eyecro.com/in-vivo/laser-induced-choroidal-neovasculariz ation-cnv/) of induced with laser reproduces the pathologic process under many DME, comprises inflammation and blood vessel occurs.EyeCRO (Oklahoma City, OK) is studied in rats.The 0th day time, 6 animals often in group carry out bilateral laser treatment to produce the damage of every eyes 3 place.When the 3rd day and the 10th day, by 3 μ g Anti-TNF-α rat IL-6 antibody (R & D Systems AF506; Minneapolis, MN) vitreum (IVT) is expelled in experimental group, and PBS or anti-vegf polyclonal antibody (R & D Systems AF564) is administered to carrier and positive controls respectively.The 15th and 22 days body interimages to measure lesion region.Reduce blood vessel the 15th day and the 22nd day anti-IL-6 treatment group significantly relative to vehicle Control to occur.Between anti-IL-6 treatment group and anti-vegf positive control, in response, (response) does not have significant difference.Fig. 1 shows the result of this experiment.These data show, IVT uses IL-6a, such as anti-IL6 antibody, can reduce blood vessel and occur to the level similar to anti-vegf positive control (anti-IL-6 to vehicle Control, at the 15th day p=0.0054 with at the 22nd day p=0.0005) in CNV rat model.

These data show, local blocks IL-6 to treatment illness in eye, such as, relate to the illness in eye of vascular leakage, if macular edema is useful.

Embodiment 2: candidate antibodies IL-6 antagonist

Use the flow process exploitation candidate antibodies IL-6 antagonist first relating to immunization.Immunization is implemented by Contract Research Organization (CRO) under the instruction of contriver.To 5 BALB/C mice subcutaneous injections containing 80 μ g people IL-6 (R & D Systems, cat#206-IL/CF, Minneapolis, MN), the PBS of 1M NaCl and freund's adjuvant.Twice booster immunization is carried out with the IL-6 of 80 μ g and 50 μ g.Splenocyte is collected and with P3x763Ag8.653 myeloma cell fusion to form hybridoma from the mouse of the highest titre.

Doma supernatant screening IL-6 is combined and Antagonism.For in conjunction with ELISA, with the PBS bag containing 1 μ g/mL people IL-6 by Costar 9018 plate, 4 DEG C are spent the night.With the PBS closed pores containing 2%BSA, washing, then often plants doma supernatant with 50 μ L and hatches, and the PBS of described doma supernatant containing 2%BSA dilutes with 1: 2.After 60 minutes, the 300 μ l PBSs of hole containing 0.1%Tween-20 are washed three times.Then every hole is incorporated in the anti-mouse HRP of 1: 3000 dilution in PBS-BSA, and hatches 30 minutes.Plate hole does washing as above, then adds 3,3 ', 5,5 '-tetramethyl benzidine (TMB) substrate, and 450 and 550nm place measurement signal.For antagonism research, HEK-Blue ^tMhatch with the people IL-6 of increasing concen-trations under the existence of the doma supernatant that-IL6 reporter cell (InvivoGen, San Diego, CA) dilutes at 1:10.After 20-24 hour, 20 μ l supernatant liquors and 180 μ l QuantiBlue ^tM(InvivoGen) mix, and measure absorbancy at 655nm place.

Study based on combination and antagonism, filter out hybridoma 64 as leading (lead) by applicant and at CRO subclone.Using enzyme-linked immunosorbent assay (ELISA) to test hybridoma 64 (mouse monoclonal) further suppresses IL-6/IL-6R α mixture to be attached to the ability of gp130.By ELISA, the hybridoma 64 of 1.5 μ g/ml concentration reduces IL-6/IL-6R α mixture significantly and is attached to immobilized gp130 (Fig. 2).

Screen described subclone again and check order by the variable domains of 5 ' RACE pcr amplification subclone 64.58.The sequence (being called m64) of mouse variable domain is as follows:

M64 VH (variable heavy chain)

QVQLQQSGAELVRPGTSVKVSCKASGYAFSNYLIEWVKQRPGQGLEWIGVITPGSGTINYNEKFKGKAVLTADKSSSTVYMQLSSLTSDDSAVYFCAKSRWDPLYYYALEYWGQGTSVTVSS(SEQ ID NO：13)

M64 VL (variable light)

DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPLTFGAGTKLELK(SEQ ID NO：14)

In order to create humanized sequence, the complementary determining region (CDR) of m64 is transplanted in people's germline framework (germline framework) similar to mouse sequence selected by computational algorithm.Humanized sequence's (being called h64) following (the residue underscore of change indicates compared with m64 sequence) and there is the identity (VH) of about 79.5% and the identity (VL) of 84.4% with mouse sequence:

h64 VH

QVQL VQSGAE VKKPG SSVKVSCKASGYAFSNYLIEWV RQ APGQGLEW MGVITPGSGTINY AQKF QG RVTITAD ES TST AYM ELSSL RS ED TAVY YCA RSRWDPLYYYALEYWGQGT TVTVSS(SEQ ID NO：15)

h64 VL

DIV MTQSP DSLAVSLG ERATI NCRASESVDNYGISFMNW YQQKPGQPPKLLIYAASNQGSGVP DRFSGSGSGTDF TL TI SSLQAED VA VY YCQQSKEVPLTFG QGTKLE IK(SEQ ID NO：16)

Synthesize humanized sequence with DNA2.0 (Menlo Park, CA), then itself and human IgG1's constant domain are merged the expression vector being cloned into pcDNA3.1 in the lump and originating.By transient transfection Freestyle ^tM-293 cells (Invitrogen, Grand Island, NY) are expressed, and by Protein A Chromatography IgG purification.In combination and antagonism research, compared with its m64 ancestors (predecessor), h64 IgG shows sizable effect to be reduced.Therefore yeast display is utilized to recover the avidity lost.

In order to implement the affinity maturation (affinity maturation) that object is recovery or raising humanization h64IgG avidity, h64 antibody sequence is cloned into produce Fab molecule in the yeast vector in pYC2/CT source again, in wherein said carrier FabH chain pass through (G4S) 3 connexon and anti-FITC scFv 4m5.3 merge.Then h64 Mutant libraries is produced by fallibility PCR (scheme according to (2006, Nature Protocols, 1:755-768) such as Chao).Express H64 variant and catch variant by the yeast being marked with FITC-PEG-NHS on surface, then hatching with biotinylated people IL-6.The IL-6 combined is detected with avidin-APC, and at BD FACSAria ^tMsorter is selected the cell being combined with maximum amount IL-6, the amount of the IL-6 wherein combined is relevant to the amount of the Fabs of displaying.Through four-wheel screening, high-affinity variant population is selected and is checked order.Cloned sequence (being called h64-1.4) selected by affinity maturation is as follows, and the sudden change (sudden change as compared with VH with the VL sequence of h64) selected in it shows with runic, and CDRs underscore indicates.These are variable domains (and IL-6a molecule of following 020 and 029) of 018.It should be noted that complete Fabs comprises the CH1 structural domain of CK and IgG1.In the context of this application, " Fab " heavy chain mentioned or light-chain amino acid sequence refer to the part of the Fab worked that the sequence that sequence can be derived by derived light chain sequence and heavy chain forms.

H64-1.4 VH (018VH) (variable domains)

H64-1.4 VL (018VL) (variable domains)

H64-1.4 variable domains is cloned in pcDNA3.1 human IgG1 carrier again, and at Freestyle ^tMthe IgG1 of total length is expressed in-HEK293 cell (Life Technologies).The IgG of the purifying obtained is obviously more efficiently than original h64 antibody in combination and the research of cell antagonism.Use Yeast system to test avidity, described avidity brings up to 43pM from the 343pM of initial humanized molecule.The effect of the antagonist using HEK-Blue cell system to measure improves about 10 times.

H64-1.4 IgG is rearranged to Fab to use in eye and other indications.In addition, another library generation taken turns and screening based on yeast is carried out, to improve avidity further.Through four-wheel screening, there is the significant enrichment of the VH variant with A76V sudden change.Comprise the antibody of A79V, variant and fragment thereof and be called 019 IL-6a antibody, variant and fragment thereof.

Embodiment 3: the selection of form

In order to study the form be applicable to of the IL-6 antagonist based on antibody, test the transient expression of the IL-6 antibody selected as previously described, stability, aggregation, binding affinity and IC50, wherein said antibody uses Fab, scFv (VH-VL) and scFv (VH-VL) form of 018 sequence.

One of them aforementioned experimental results of candidate IL-6a molecule (comprising the sequence of 018 variable region) is shown in table 1.

Table 1

The qualification of these data displays is based on the method for the various forms of key features of IL-6 antagonist antibodies, and illustrate for the IL-6 antagonist comprising 018 variable region, 018Fab form, compared with scFv structure, has most popular feature in maximum crucial classifications (as expression, stability, gathering and binding affinity).The IC50 of 018Fab drops in the rational scope that is used for the treatment of.

The example of embodiment 4:IL-6a antibody, fragment and derivative

Use method described in the invention, applicant has identified following sequence.The sequence of underscore represents the CDRs of heavy chain and light chain.Other sequence can find in whole specification sheets.

018 heavy chain (total length in IgG1 framework; Fl018HC) peptide sequence

QVQLVQSGAEVKKPGSSVKVSCKAS GYALSNYLIEWVRQAPGQGLEWMG VITPGSGTINYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR SR WDPLYYYALEYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：19)

018 heavy chain (total length in IgG1 framework; Fl018HC) nucleotide sequence

CAAGTGCAGCTGGTGCAGTCAGGGGCCGAGGTTAAGAAGCCAGGGAGCAGCGTCAAGGTATCTTGTAAAGCGTCTGGTTACGCCCTTTCAAACTACCTGATCGAATGGGTGAGGCAGGCTCCCGGCCAAGGCCTGGAATGGATGGGAGTTATCACCCCTGGGAGCGGCACCATTAATTACGCCCAGAAATTTCAGGGACGAGTGACGATTACCGCCGACGAGTCCACCAGTACTGCCTACATGGAGCTGTCCTCACTCCGCAGCGAGGACACGGCAGTTTACTACTGCGCCCGGAGTCGATGGGACCCTCTTTACTATTATGCTCTGGAATACTGGGGCCAGGGAACGACCGTTACAGTGTCATCTGCTAGCACAAAAGGACCATCAGTCTTCCCACTTGCTCCTTCATCTAAGAGCACAAGTGGTGGCACTGCAGCCCTTGGCTGCCTGGTGAAAGATTATTTCCCCGAACCTGTTACAGTTTCTTGGAACTCCGGTGCACTGACATCCGGAGTACACACTTTCCCAGCTGTGCTGCAGAGCTCAGGACTGTATAGCCTGTCTTCGGTGGTCACTGTTCCATCGTCGAGTCTTGGCACACAGACATATATTTGCAACGTCAATCACAAGCCCTCCAACACAAAAGTGGATAAGAAGGTCGAGCCCAAATCTTGTGACAAGACCCATACGTGTCCTCCCTGTCCCGCCCCTGAACTGCTGGGAGGCCCTTCTGTGTTCCTGTTCCCACCTAAGCCAAAGGACACTCTGATGATCAGCCGGACTCCCGAGGTTACCTGTGTGGTGGTGGATGTGTCTCATGAAGACCCTGAGGTTAAGTTCAATTGGTACGTGGATGGCGTCGAGGTGCATAACGCAAAAACCAAGCCGAGAGAGGAGCAGTACaatAGCACCTATAGAGTAGTGAGCGTCCTGACTGTCTTACATCAGGATTGGCTCAATGGTAAAGAATATAAGTGCAAGGTAAGCAACAAGGCCCTACCCGCACCAATAGAGAAGACCATCTCCAAGGCGAAAGGTCAGCCCAGGGAGCCCCAGGTTTATACACTGCCTCCCTCACGCGACGAATTAACAAAGAATCAGGTGTCTCTCACCTGTCTCGTCAAGGGCTTTTACCCTTCCGACATCGCCGTGGAGTGGGAATCCAATGGCCAGCCTGAGAACAATTATAAGACAACTCCCCCAGTCCTGGATTCAGATGGGTCGTTCTTTCTATATAGTAAGTTGACCGTGGATAAGTCTCGCTGGCAACAGGGGAACGTGTTCTCTTGCTCTGTTATGCATGAAGCGCTGCACAATCATTATACCCAGAAGTCCCTGTCCCTGAGCCCCGGGAAG(SEQ ID NO：20)

018 Fab heavy chain (018FabHC) peptide sequence in IgG1 framework.CDRs indicates with underscore

QVQLVQSGAEVKKPGSSVKVSCKAS GYALSNYLIEWVRQAPGQGLEWMG VITPGSGTINYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR SR WDPLYYYALEYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC(SEQ ID NO：1)

018 full-length light chains (fl018LC) peptide sequence.CDRs indicates with underscore

This is also the light chain of 020 and 029 IL-6 antagonist

018 full-length light chains (018LC) nucleotide sequence in IgG1 framework

019 Fab heavy chain (019FabHC, identical with 018FabHC sequence except A79V (runic/italic)

019 VH (variable region/019HC, identical with 018HC variable region sequences except A79V (runic/italic)

019 light chain of antibody (019LC) sequence (polypeptide and nucleic acid) is identical with 018LC

CDR1 (VH CDR1 018): GYALSNYLIE (the SEQ ID NO:4) of 018HC

CDR2 (VH CDR2 018): VITPGSGTIN (the SEQ ID NO:5) of 018HC

CDR3 (VH CDR3 018): SRWDPLYYYALEY (the SEQ ID NO:6) of 018HC

CDR1 (VL CDR1): RASESVDNYGIPFMN (the SEQ ID NO:7) of 018LC

CDR2 (VL CDR2): AASNRGS (the SEQ ID NO:8) of 018LC

CDR3 (VL CDR3): QQSEEVPLT (the SEQ ID NO:9) of 018LC

CDR1 (VH CDR1 019): GYALSNYLIE (the SEQ ID NO:4) of 019HC

CDR2 (VH CDR2 019): VITPGSGTIN (the SEQ ID NO:5) of 019HC

CDR3 (VH CDR3 019): SRWDPLYYYALEY (the SEQ ID NO:6) of 019HC

Embodiment 5: epi-position and structure mapping

Epitope mapping

Functional epitope mapping is carried out to selected candidate IL-6 antagonist.Find that candidate antibodies (mouse 64 antibody) does not weaken the combination of IL-6R α to IL-6 in ELISA, shows this candidate antibodies not binding site I.The experiment additionally carried out proves to show that gomphosis mouse 64 antibody weakens IL-6/IL-6R α mixture to the combination of gp130 in ELISA, illustrates that the site II of IL-6 or site III carries the binding site of this antibody.Also find that mouse 64 antibody does not significantly block known site III binding antibody AH-65 (Immunotech, Marseille, France) and the combination of IL-6, illustrates the site II of this candidate antibodies in conjunction with IL-6.These data show to produce the antibody for site II, and demonstrate the method for this antibody of qualification.

In order to define epi-position further, in yeast, create the sudden change of IL-6, itself and 4m5.3 (Boder et al., 2000, Proc Natl Acad Sci USA 97,10701-10705; Chao et al., 2006, Nat Protoc 1,755-768) merge mutually.Expressed mutant is the mutant in people IL-6 with following single or two sudden change: R24E/D27E, R30E, Y31E, D34R, S118R/V121E, W157E, Q159E/T162P, K171E and R179E.The IL-6 molecule of the sudden change of described expression is used to the binding with 018 (Fab).Observe the reduction of R24E/K27E, Y31E, D34R and S118R/V121R avidity of 018 (FAB), it is all arranged in the site II of IL-6.Therefore, invention described in the invention comprises antibody, and it can be attached at least one in people IL-6 position 24,27,31,34,118 and 121, two, three, four, five or six amino acid, maybe can in conjunction with the equivalent site in IL-6.

The organization definition of site II epi-position

Calculate lower column distance structurally to define site II.This calculating based on the crystalline structure of IL-6/IL-6 α/gp130 six aggressiveness, PDB 1P9M (Boulanger et al., 2003, Science 300:2101-2104).The spiral 1 of IL-6 is positioned between (runs) site I and site II, causes some residue close to site II but has the side chain pointing to site I, as R30.D2 and D3 refers to the extracellular domain of IL-6R α.

The amino acid of following IL-6 is confirmed as falling into gp130-D2-D3's within: L19, R24, K27, Q28, R30, Y31, D34, E110, Q111, R113, A114, M117, S118, V121, Q124, F125 and K128.

Within the amino acid of following IL-6 is confirmed as falling into the 7A of gp130-D2-D3: L19, E23, R24, I25, K27, Q28, I29, R30, Y31, D34, K41, Q102, E109, E110, Q111, A112, R113, A114, V115, Q116, M117, S118, K120, V121, L122, Q124, F125 and K128.

Therefore, it is possible in conjunction with the amino acid whose molecule of one or more IL-6 decline angle of striking II 5A or 7A, as antibody or its fragment, can be IL-6a.

There is provided people IL-6 sequence below as a reference (sequence that underscore indicates is leader sequence).Amino acid italic within the 7A of gp130-D2-D3 marks.Amino acid number, as the sudden change for defining epi-position, not containing leader sequence.

People IL-6

Carry out the Fab fragment of experiment test h64-1.4 humanized antibody, and prove that this fragment is due to target site II, thus IL-6 cis and upside down signal can be blocked.The existence of solubility IL-6 acceptor (sIL-6R) does not change the effect of Fab fragment.This is contrary with anti-IL-6R IgG, and its effect under the existence of sIL6R decreases, and it only blocks cis signal.

These experiments prove the antibody of target site II or the fragment of this antibody, as Fab fragment can be used for the cis and the upside down signal that suppress IL-6.

Embodiment 6: primate study

Because non-primate can have very big-difference with the vigor of primate (activities), therefore generally use PK and other parameters of the further evaluate candidate IL-6 antagonist of non-human primate.People IL-6 has the different of 7 sites from cynomolgus monkey and macaque IL-6, and one of them is positioned at site (amino acid 28) II and identical with the site II of cercopithecus aethiops IL-6.This antibody seeming only to reduce containing 018 sequence is about 3-4 binding ability doubly.The ability being attached to non-human primate IL-6 is the useful feature of of IL-6 antagonist, and it promotes the exploitation as the material standed for of medicine, as by making the test in non-primate, as toxotest becomes possibility.

As most of IL-6 antibody, because the low sequence homology of IL-6, there is not cross reaction with the IL-6 of rodent, rabbit or dog in anti-IL-6 antibodies described in the invention.But, in avidity research, find that 018Fab is similar to the IL-6 (table 2) of people's avidity in conjunction with cynomolgus monkey and cercopithecus aethiops.

Table 2: to the unit price avidity (018 Fab) of the various IL-6 of various species

These data further demonstrate the ability of IL-6a specific binding described in the invention and in the animal be applicable to, form the ability with the molecule allowing the feature detecting (as toxicology and reproductive study).

Embodiment 7: the expression improving IL-6a

In order to improve the expression of the Fab polypeptide of 018Fab and 019, means known in the art are utilized to prepare the construct introducing 5 additional amino acids (DKTHT) to heavy chain CH1/ hinge area.The sequence of altered 018Fab heavy chain is shown in SEQ ID NO:24 below.018 sequence changed is called 020 in the present invention and 019 sequence changed is called 021 in the present invention.020 molecule (020Fab heavy chain and 018Fab light chain) with there is 018Fab heavy chain compare the expression with improvement with the parent Fab of 018Fab light chain.019 molecule does not show significant avidity difference compared with 020 molecule.The expression of 020 and 019 adds about 2 times, the impact that avidity is not changed respectively.

020 heavy chain (there is at C-terminal the Fab of DKTHT))

Use 020Fab at HEK-Blue ^tMthe antagonistic action of IL-6 is measured in IL-6 recipient cell (InvivoGen, San Diego, CA).At 020 or IL-6R Alpha antibodies (Cell Sciences containing 10pM IL-6 and various concentration, Canton, MA) incubated cell in mixed solution (contain or do not contain 50nM IL-6R α), after hatching 20-24 hour, 20 μ L cell culture supernatants and 180 μ L QuantiBlue ^tM(InvivoGen) substrate mixes and hatches 1 hour; Absorbancy is measured at 655nm place.Fig. 3 A and Fig. 3 B shows the data of these experiments, demonstrates in existence or under lacking IL-6R, the ability of 020 suppression IL-6 activity.

Embodiment 8:IgG2 IL-6 antibody

Means known in the art are used to enter in human IgG2's isotype framework 018 reconstruct (reformat) to reduce the combination of Fc γ R and reduce ADCC compared with the antibody of IgG1 form.In addition, expect that 018 is reconstructed into total length form (as IgG2) and reduces due to the size of molecule from the clearance rate vitreum.

Structure/purifying anti-IL-6 IgG2 antibody

In order to use anti-IL-6 sequence construct human IgG2 antibody as previously described, from cDNA, pcr amplification goes out respectively in the human IgG2 constant domain of N-and C-end with NheI and MluI restriction site.Purified pcr product, with NheI and MluI digestions, then connect in pTT5 carrier, described carrier comprises anti-IL6 variable domains, as SEQ ID NO:1 (seeing above).Obtain the IgG2 sequence of heavy chain of total length like this.Plasmid containing the full-length light chains comprising 018 sequence is used to provide light chain.

In order to therefore the combination reducing FcRn further also reduces the recirculation of IL-6a, in heavy chain, carry out point mutation.Use mutagenesis (Agilent Technologies, Santa Clara, CA) manufactures sudden change.Use poly-(ethyleneimine) (PEI) by heavy chain and light chain plasmids cotransfection in the instantaneous culture of 100mL HEK293-6E cell, and cultivate to allow the expression carrying out about 5 days.The antibody of this generation contains site II bound fraction and the IgG2 structure of anti-IL-6.This structure containing 018CDRs is called 018IgG2 or 029 in the present invention.Suddenly change at residue I253 place manufacturing place.

Described IgG2 molecule is expressed well and blocked IL-6 with the effect slightly improved compared with 020Fab in cytologic experiment.

029 mature sequence (CDRs underscore indicates)

029 heavy chain

029 light chain

The FcRn changed combines

IL-6 can have some positive systematic effect.Therefore transform IL-6 to make it in vitreum, have good delay but there is the limited whole body transformation period to be favourable.Reduce or eliminate FcRn to combine and should reduce any systematicness escaped into the medicine of circulation and accumulate, thus the security of raising IL-6a.

Therefore, transport due to FcRn mediation may increase antibody and flow out from eye, therefore by introducing sudden change (see SEQ ID NO:23) numbering according to Martin etc. at residue I254, H311 or H436 of Fc, Molecular Cell, 7:4,867-877 (2001)), modify 020IgG2 further to eliminate the combination of FcRn.The site of sudden change is indicated with runic in SEQ ID NO:23; I254 sports A or R; H311 sports A or E, H311 sports N and D313 sports T, and H436 sports A, and (after leader sequence open numbering, described leader sequence is indicated with underscore in SEQ ID NO:23.IL-6 antagonist containing this sequence is called 018IgG2m.

Anti-IL-6 heavy chain (IgG2) (regular font: VH; Italic: CH) (leader) show mutational site (runic)

Anti-IL-6 heavy chain (IgG2) (regular font: VH; Italic: CH) there is leader sequence (underscore) to show mutational site (runic)

Therefore, some embodiments comprise the antibody with sequence of heavy chain, described sequence of heavy chain is described in SEQ ID NO:23, and have I254 (as A or R), H311 (sporting A or E), H436 (sporting A), or D313 (sporting T) and H311 sport the sudden change of N.

SEQ ID NO:25 is because herein is provided a kind of sequence, when at I133 (as I133A or I133R), H190 (as H190A or H190E), H315 (H315A), or D192 and H190 (as D192T and H190N) place is when undergoing mutation, this sequence can be used for antibody, fragment or derivatives thereof has the polypeptide weakening Fc and combine under (as pH5.5) or lysosomal pH at a low ph to produce, and/or produce with parent or other containing this sequence reference molecule compared with there is the polypeptide of the whole body transformation period of weakening.

Anti-IL-6 light chain (IgG2) (regular font: VH; Italic: CH)

Embodiment 9: preparation stability

Test the stability of anti-IL-6/IgG1 Fab fragment (comprising IgG1 CH1 structural domain), described test determines initial Tm in PBS, the Tm then in a series of damping fluid and vehicle by using differential scanning fluorescence.Find that Tm is increased to exceed 80 DEG C by citrate buffer (pH5.5).Therefore, in some embodiments, IL-6a to be provided in citrate buffer and to have the Tm of at least 80 DEG C in some cases.

Use SEC-MALS tests aggregation extent and 20mg/ml does not observe gathering in phosphate buffered saline buffer (PBS).

Embodiment 10: be suitable for the antibody to pH sensitivity strengthening PK

IL-6 can have some positive systematic effect.Therefore transform IL-6 to make it in vitreum, have good delay but there is the limited whole body transformation period to be favourable.Reduce or eliminate can to reduce with the combination of FcRn and anyly escape medicine into circulation in the accumulation of whole body, thus improve the security of IL-6a.Therefore, transport due to FcRn mediation may increase antibody and flow out from eye, therefore (number according to Martin etc. by introducing sudden change at residue L254, H311 or H435 of Fc, Molecular Cell, 7:4,867-877 (2001)), to modify 020IgG2 further to weaken the combination of FcRn.This antibody is called IL-6pH antibody or anti-IL-6pH in the present invention and is further described below.

The generation of IL-6ipH antibody

The pKa of Histidine is about 6.0, and the Histidine inserted on bonding interface can turn into rear destruction combination at side chain protons when low pH.Use anti-IL-6 site as described in the present invention II targeting antibodies, from 018, produce the library that comprises the CDR variant being rich in Histidine, and use yeast display from this library, screen the combination of pH sensitivity.The library produced is the combinatorial library with CDRs, and described CDR is encoded by degenerate codon, makes each residue be a wildtype residues (that is, identical with parental antibody), or histidine residues.Combine by oscillation sorting height under physiological pH (7.4) and bend down at endosome pH (5.5) and be incorporated into row filter.

Identified the mutant of a yeast-selection, it has combination relatively high under pH7.4 (the unit price Kd that this mutant has a 407pM compares the Kd of the 192pM of parent) and combination relatively low under pH5.5 (the unit price Kd that this mutant has a 2.362nM compares the Kd of the 195pM of parent).This is formed in the change of avidity about 5.8 times under pH5.5.This mutant is included in multiple Histidine mutagenesis at light chain CDR1 place.Therefore, this mutant demonstrates binding ability similar to parent molecules under pH7.4, and the remarkable forfeiture of avidity under pH5.5.By methods known in the art, use this observation of ELISA, FACS and SPR analysis verification.

These data show, the IL-6a based on antibody can be created, it has the feature of the anti-IL-6 of the site II of target IL-6, can be used for suppressing the cis of IL-6 and trans activity, and have with parental antibody or other, there is wild-type Fc structural domain antibody compared with the PK of increase, the combination at least partly by changing under pH5.5 realizes.

Other embodiments are within the scope of appended claims.

Accompanying drawing is sketched

Fig. 1 is the chart of illustrative experiment result, and this experiment IVT in rat CNV model uses anti-IL-6 antibodies.VEGF antibody is applied as positive control and negative control is carrier itself.Anti-IL-6 to vehicle Control, p=0.0054 when the 15th day, p=0.0005 when the 22nd day.

Fig. 2 illustrates the chart of test mouse 64 antibody suppression IL-6/IL-6R in conjunction with the Binding experiment result of the ability of gp130.

Fig. 3 A is the chart of illustrative experiment, tests 020 blocks IL-6 intracellular signaling (signaling) ability when lacking too much solubility IL-6R α in this experiment.Experiment carries out on HEK-Blue-IL-6 cell having in 0.2ng/mL IL-6 and 2 μ g/mL IL6R α situations.

Fig. 3 B is the chart of illustrative experiment, tests 020 and lack the ability of depositing and blocking IL-6 intracellular signaling in case at too much solubility IL-6R α in this experiment.Experiment carries out on HEK-Blue-IL-6 cell when having 0.2ng/mL IL-6 and 2 μ g/mL IL6R α.

Detailed Description Of The Invention

Imply that IL-6 works in the various diseases of such as rheumatoid arthritis, and be reported in the various diseases comprising eye disease and significantly raise.IL-6 works by cis and trans mechanism.In cis mechanism, think that free IL-6 is bonded to film mating type IL-6 acceptor (IL-6R, also referred to as IL-6R α and CD126), and then, IL-6/IL-6R mixture and gp130 (also referred to as CD130, oncostatin M acceptor, IL-6Rbeta and IL-6 signal transducer) interact, to activate the intracellular signaling in the cell containing described mixture.In trans mechanism, free IL-6 is in conjunction with solubility IL-6 acceptor (sIL-6R).IL-6/sIL-6R mixture then can in conjunction with the gp130 be present on cytolemma.Difference crucial between these mechanism is that the cell type of expressing gp130 is more than the cell type of expressing IL-6R (its expression is more limited).Therefore suppress in hope in the disease of IL-6 intracellular signaling, such as, wish to suppress widely, in the disease of IL-6 intracellular signaling, to suppress cis and trans IL-6 signal to be useful at those.Applicant has prepared IL-6 antagonist, and as anti-IL-6 antibodies, fragment and derivative, this antagonist can suppress the cis of IL-6 and trans intracellular signaling.In addition, applicant has prepared this IL-6 antagonist to realize vitreum reservation and the whole body removing faster of enhancing.

The feature of IL-6 antagonist (IL-6a)

Generally speaking, the IL-6 antagonist (IL-6a) that the present invention describes is specifically in conjunction with the site II (site 2) of IL-6, and the treatment of the illness in eye of being correlated with for IL-6 and some other diseases is useful.The illness in eye that IL-6 is relevant is a kind of disease, and wherein the biologic activity of undesirable symptom or disease is with the expression of IL-6 or exist relevant.IL-6a has high-affinity to free with the IL-6 combined in some embodiments, metastable in body, can suppress to be attached to the IL-6 (being called as IL-6/IL-6R mixture or IL-6/IL-6R in the present invention) of IL-6R and the combination of gp130, and can result for the treatment of be had.In general, IL-6a is antibody or derived from antibody.Such as, IL-6a is the humanized Fab of high-affinity, and it also effectively can stop cis and trans IL-6 intracellular signaling in conjunction with the site II of IL-6 specifically.In another example, IL-6a is full length antibody, as IgG1 or IgG2 antibody.

In some embodiments, Fab is also set to the sequence of Fc-through engineering approaches or is arranged in the antibody of total length.In some embodiments, the IL-6a (Fab as Fc-through engineering approaches) of Fc-through engineering approaches is compared with suitable contrast, as compared with the corresponding antibody without the Fc of through engineering approaches, its fragment or derivative, there is the vitreum residence time and/or the whole body removing faster of increase.These and other features of IL-6a are further described in the present invention.

Applicant designed selectively in conjunction with the IL-6 antagonist of the site II of IL-6 to provide the extensive suppression of IL-6 intracellular signaling because this quasi-molecule can suppress the combination of gp130 and IL-6, no matter IL-6 is binding film IL-6R or in conjunction with sIL-6R.In addition, target part (IL-6) toxicity (the cell-mediated cytotoxicity of antibody-dependant) that receptor-mediated removing can be avoided relative to IL-6 acceptor and be brought by ADCC.Because IL-6 plays pathology and protective effect in disease, therefore use IL-6 antagonist (IL-6a) the treatment disease relevant to the IL-6 increased can improve the situation of some aspect, but also may cause significant side effect, as systematic effect.The duality (ability of as likely in tool and/or undesirable effect) of this IL-6 path makes it be not suitable as general inhibitor for treating IL-6 associated conditions.Therefore, composition provided by the invention and method are useful for treatment, described composition suppresses the activity of at least one IL-6 but does not have the positive activity of IL-6 to affect improperly, and part is because described composition can be configured to local delivery, as being formulated as local delivery to eyes.Such as, in some aspects, IL-6a is designed to be suitable the size being delivered to specific site.In some embodiments, IL-6a is full length antibody.In some embodiments, IL-6a is in a kind ofly can has longer dwelling period in the vitreum of eyes and limited systematicness spills the mode of (systemic leakage) derived from antibody.In some embodiments, IL-6a is the antibody (as having the antibody of Fc structural domain of modification) modified, and compared with the antibody of corresponding unmodified, it in the vitreum of eyes, have longer dwelling period and/or more limited general spills.IL-6a is IgG2 antibody in some embodiments.

In some respects, IL-6a is relatively little IL-6a, as namely the fragment of antibody or other antibody derivatives are less than full length antibody, such as, derived from the Fab of IL-6 antibody.In some cases, IL-6a be in a kind of can from one tissue a part be delivered to another tissue and there is dynamic (dynamical) form of increase compared with corresponding total length IL-6 antibody.In some embodiments, IL-6a is engineered to more macromolecular Fab, itself and Fab separately compared with more may have the stop of prolongation in the region be delivered, such as IL-6a is by Fc structural domain dimerization.In some embodiments, Fc structural domain is by through engineering approaches thus make Fc part have (ablated) that slacken or the FcRn weakened combines, thus the local residence that extends can be caused, stop and reduce whole body accumulate as increased vitreum compared with the identical IL-6 combination (IL-6 binding entity) comprising wild-type Fc.

IL-6 antagonist described in the invention also has sufficiently high affinity to its target IL-6 thus effectively improves at least one undesirable effect of IL-6.Described inhibitor or sufficiently stable to contribute to it as therapeutical agent.

Generally speaking, the PK of IL-6a, as the IL-6a be suitable for use in eyes has the PK being enough to provide result for the treatment of in the site of sending (as vitreum).In infinite example, described PK can be the transformation period of at least 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 8 days, 10 days, 14 days, 21 days, 28 days or 30 days.

Be attached to the qualification of the IL-6 antagonist of site II

In general, any method as known in the art all can be used for producing can in conjunction with the molecule of IL-6, and such as, in experiment, polypeptide libraries or molecular library can be used for screening and have the polypeptide of the ability being attached to IL-6 or the candidate compound of compound.Once identify this candidate compound, the binding site of method deterministic compound as known in the art can be used.Such as, the ability of wild-type IL-6 can be attached to and this bonding force and this molecule made comparisons with the binding ability of the IL-6 suddenlyd change at site I, site II or site III by test molecule.In embodiments, IL-6a of the present invention retains the ability being attached to IL-6/IL-6R α mixture and being attached to IL-6, and stops IL-6/IL-6R α to be attached to gp130.In embodiments, such as IL-6a of the present invention by the combination of the site II with IL-6 can with gp130 competition binding IL-6/IL-6R α mixture.Methods known in the art can be used to measure this binding ability.

Such as, HEK-Blue can be used ^tMiL-6 Analytical system (InvivoGen, San Diego) test I L-6a material standed for.HEK-Blue ^tMiL-6 cell people IL-6R and STAT3-that be stable transfection can induce the HEK293 cell of SEAP reporter gene.Under IL-6 exists, STAT3 is activated and SEAP is secreted.Such as QUANTI-Blue can be used ^tM(InvivoGen, San Diego) measures SEAP.Add IL-6 antagonist to cell and inhibit free and soluble receptors and IL-6 combination, thus prevent the secretion of SEAP or reduce the level of SEAP.

K _drefer to specific antibodies-AI or antibody fragment-AI in conjunction with the affine equilibrium constant.Work as K _dless than or equal to 250pM, as during less than or equal to 225pM, 220pM, 210pM, 205pM, 150pM, 100pM, 50pM, 20pM, 10pM or 1pM, antibody or its fragment are considered to specificity and are combined with antigen.Method well known in the prior art can be used to determine K _d, such as surperficial plasmon resonance, as used BiaCore ^tMsystem determines K _d.

K _offrefer to the dissociation rate constant of specific antibodies-AI or antibody fragment-antigenic compound.Surperficial plasmon can be used to resonate, such as, use BiaCore ^tMsystem determination dissociation rate constant.A relatively slow K _offthe feature desired by treatment can be produced, as allowed to use inhibitor with low frequency to needing the individuality of this treatment.

Specificity

The feature of IL-6 antagonist disclosed in this invention relates to their binding specificity.As discussed above, IL-6 can be used as free IL-6 and exists as the IL-6 being attached to solubility IL-6R α.Applicant identifies, and compared with the inhibitor in conjunction with IL-6 site I, the site II of IL-6 is the best target of IL-6 antagonist.The inhibitor of site I can suppress the combination of free IL-6 and IL-6R α.But this inhibitor can not stop the activity caused by the mixture existed in advance, except by replacement, it is limited to the K of IL-6/IL-6R mixture _off.In another possibility, the inhibitor in conjunction with IL-6R α is not too suitable, unless because it exists with saturation concentration, this inhibitor has the ability of limited prevention IL-6 activity.Because the amount of IL-6 acceptor is normally quite high compared with the amount of IL-6, therefore this method may need to use less desirable in a large number by suppressing the compound of IL-6 activity with receptors bind.In embodiments, IL-6 antagonist described in the invention (antibody as described in the present invention and fragment and derivative) thereof even can block the activity of IL-6 when IL-6 is attached to IL-6R.Therefore, the advantage of IL-6a described in the invention is compared with the inhibitor of target IL-6 acceptor, and the amount realizing the compound used needed for result for the treatment of is relatively little.Antireceptor antibody has been in the news and has been removed fast by receptor-mediated removing, This significantly limits the P of antibody _k, therefore need escalated dose, more high frequency administration or use aforementioned both.In addition, the problem of antireceptor antibody and anti-site I IL-6 antibody is that they significantly increase the tissue concentration of IL-6 by the removing approach that the normal subject of block ligand mediates, thus makes experimenter be in the potential unwanted level of IL-6 in tissue.In addition, near the site using the inhibitor of target IL-6R α that inhibitor may be made to be present in seek inhibitor and undesirable site (as whole body therapeutic).Use the IL-6 allowing with the IL-6a that site II (gp130 is in conjunction with this site) combines to suppress free IL-6 and combine IL-6R, but do not activate the IL-6 approach through gp130.Therefore, IL-6 antagonist described in the invention is designed to the IL-6 (solvable and be attached to acceptor) in conjunction with two kinds of forms, and be particularly attached to the antagonist of the site II of IL-6, this antagonist can in conjunction with the IL-6 of two kinds of forms.The described in the invention composition comprising IL-6a suppresses cis and upside down signal by IL-6.

In some cases, Compounds and methods for provided by the present invention is designed to provide a kind of effective IL-6 blocker, and it is enough to sign or the symptom for the treatment of at least one IL-6 associated conditions, occurs and/or inflammation as suppressed blood vessel.

Compound described in the invention also can be used for treating the illness in eye being feature with less desirable high-level IL-6, if the disease in vitreum is (see Yuuki et al., J Diabetes Compl 15; 257 (2001); Funatsu et al., Ophthalmology 110:1690, (2003); Oh et al., Curr Eye Res 35:1116 (2010); Noma et al., Eye 22:42 (2008); Kawashima et al., Jpn J Ophthalmol 51:100 (2007); Kauffman et al., Invest Ophthalmol Vis Sci 35:900 (1994); Miao et al., Molec Vis 18:574 (2012)).

Usually, IL-6a described in the invention is IL-6 intracellular signaling potent antagonist.This part ground is realized the molecule that IL-6 has high-affinity by design, and such as, in using the HEK-Blue IL-6 of 10pM IL-6 to test, IC50 is less than or equal to 100pM.Can according to the K of IL-6a _ddetermine the high-affinity of IL-6a, such as, be less than or equal to the K of 1nM _d, be less than or equal to the K of 500pM _d, be less than or equal to the K of 400pM _d, be less than or equal to the K of 300pM _d, be less than or equal to the K of 240pM _dor be less than or equal to the K of 200pM _d.

Express or the biotechnological formulation IL-6a (such as albumen or polypeptide are as antibody, fragment or derivatives thereof) of active related disorders with the IL-6 increased to prepare to can be used for treating, preferably described biotechnological formulation IL-6a has high productive rate usually.Such as, suitable productive rate is greater than or equal to 1g/L (as greater than or equal to 2g/L, greater than or equal to 5g/L or greater than or equal to 10g/L).

In order to effectively use IL-6 antagonist, inhibitor is needed to have the solubleness matched with the concentration that will use.Such as, when the IL-6a of full length antibody, its solubleness is more than or equal to 20mg/ml, is more than or equal to 10mg/ml, is more than or equal to 5mg/ml or is more than or equal to 1mg/ml.

In addition, inhibitor must have high stability under the body temperature sent and avtive spot and stability in storage thus become a kind of feasible treatment.Such as greater than or equal to the T of 60 DEG C _m(as greater than or equal to 60 DEG C, greater than or equal to 62.5 DEG C, greater than or equal to 65 DEG C, greater than or equal to 70 DEG C, greater than or equal to 73 DEG C, greater than or equal to 75 DEG C) with greater than or equal to the T of 45 DEG C _onset, as greater than or equal to 50 DEG C, greater than or equal to 51 DEG C, greater than or equal to 55 DEG C or greater than or equal to 60 DEG C.T can be determined by methods known in the art _mand T _onsetmethod.

The antagonist with required characteristic can be selected from the type of molecule as known in the art, such as antibody, comprise fragment and the derivative of the antibody of the site II of target IL-6, it generally retains or maintains the feature (binding characteristics as needs) of enough parent IL-6 antibody.This antagonist comprises the Fab fragment containing Fc part of Fab fragment, scFvs, through engineering approaches, and the full length antibody with the site II targeting antibodies framework being different from parent IL-6 of through engineering approaches.

In certain aspects, IL-6a described in the invention comprises people's antibody antigen-binding site, and this site can be competed or cross competition can in conjunction with the antibody of the site II of IL-6 or its fragment.Such as, antibody or its fragment can be made up of the structural domain of VH disclosed in the present invention and VL structural domain, and VH and VL structural domain comprises one group of CDRs of the site of IL-6/ disclosed in the present invention II binding antibody.

Any suitable method can be used, such as, determine the structural domain that is combined with IL-6a and/or epi-position by the multiple site on sudden change IL-6.Those epi-positions containing sudden change stop or reduce the combination of IL-6a, and IL-6 part is participated in directly with the combination of IL-6a or as the configuration remote effect binding site by affecting IL-6.Additive method can be used to determine the amino acid that IL-6a combines.Such as, peptide-combination scanning can be used, as the enzyme linked immune assay (ELISA) based on PEPSCAN.In the scanning of such peptide-combination, the short overlapping peptide being derived from antigen is incorporated into row filter systematically for itself and binding members.Described peptide covalently can be connected to support surface, to form peptide array.Peptide can be linear or limited conformation.The polypeptide that the end that can be used in each peptide sequence has halfcystine (cys) residue produces limited conformation.Can peptide be made to support surface to keep annular conformation direct or indirect for cys residue covalent coupling.Therefore, the peptide used in described method may have the cys residue that is added on each end of the peptide relevant to antigen fragment.Also can use double-doughnut peptide, wherein cys residue is positioned or extraly near the middle part of peptide sequence.Can peptide be made to support surface to form the conformation in every side of central cys residue with a ring of dicyclo direct or indirect for cys residue covalent coupling.Generation peptide can be synthesized, thus can modify cys residue in the position needed, although it is not the natural generation of site II sequence at IL-6.Optionally, linear or that conformation is limited peptide can be screened in peptide-binding tests.Peptide-the peptide that qualification (as used ELISA) a group is attached to binding members can be comprised in conjunction with scanning, wherein said peptide has the aminoacid sequence corresponding with the fragment of IL-6a (as comprising the peptide of about 5,10 or 15 continuous residues of IL-6a), and peptide described in comparison is to determine the footprint of the residue that binding members combines, wherein this footprint comprises the common residue of overlapping peptide.Alternatively or extraly, peptide-combination scanning method can be used with at least selected signal: noise ratio qualification IL-6a will in conjunction with peptide.

The residue that additive method determination antibody as known in the art combines can be used, and/or confirmation peptide-in conjunction with scanning result, comprise such as, rite-directed mutagenesis (as described in the present invention), hydrogen deuterium exchange, mass spectrum, NMR and X-ray-crystallography method.

Usually, useful IL-6a described in the invention is human antibody molecules, Humanized antibody molecules or its binding fragment.Usually, antibody is monoclonal antibody.The source of this antibody can be people, mouse, rat, camel, rabbit, sheep, pig or ox, and can produce described antibody according to those methods as known in the art.

Usually, IL-6a at least comprises the CDR of antibody, and described CDR is specifically in conjunction with the site II (as people IL-6) of IL-6.Structure with CDR of the present invention or group CDRs can be heavy chain of antibody or sequence of light chain or its substantial portions, in heavy chain and light chain CDR or group CDRs be positioned at natural generation by the correspondence position of CDR or the group CDRs in VH and the VL antibody variable territory of rearranged immunoglobulin genes encoding.By people such as reference Kabat, structure and the position in immunoglobulin variable domain territory are determined in 1983 (National Institutes of Health) and the renewal (updates) using any internet search engine to find under " Kabat ".

IL-6a as antibody generally comprises antibody VH domain (as SEQ ID NO:1 or SEQ ID NO:3) and/or VL structural domain (as SEQ ID NO:2).VH structural domain comprises one group of heavy chain CDRs (VHCDRs), and VL structural domain comprises one group of light chain CDRs (VLCDRs).Provide the example of this CDRs in embodiment 3, the example of this CDRs is described in SEQ ID NOs:4-9.Antibody molecule can comprise the antibody VH domain that contains VHCDR1, VHCDR2 and VHCDR3 and framework.It comprises the antibody VL domain containing VLCDR1, VLCDR2 and VLCDR3 and framework alternatively or also.

IL-6 antagonist disclosed in this invention comprise as those VHCDR1 and/or VHCDR2 disclosed in the present invention and/or VHCDR3 and/as those VLCDR1 and/or VLCDR2 disclosed in the present invention and/or VLCDR3.IL-6a can comprise the CDRs of one or more any antibody, fragment or derivative described in the invention.IL-6a can comprise one group of VHCDRs (as VHCDR1, VHCDR2 and VHCDR3), and it can also comprise one group of VLCDRs (as VLCDR1, VLCDR2 and VLCDR3) alternatively.CDRs can come from one or more antibody described in the invention, fragment or derivatives.Such as, VLCDRs can come from the antibody identical or different with VHCDRs.

Usually, VH structural domain and VL structural domain match to provide an antibody antigen-binding site.The HC structural domain of such as SEQ ID NO:1 or SEQ ID NO:3 and the LC structural domain of SEQ ID NO:2 match.In some cases, VH or VL structural domain can be used as IL-6a alone.

In some aspects, IL-6a is antibody molecule, fragment or derivatives thereof, it comprises the VH domain sequence that (i) and VH structural domain described in the invention (such as the VH structural domain of SEQ ID NO:1 or SEQ ID NO:3) have at least 60,70,80,85,90,95,98 or 99% amino acid sequence identity, or one group of VHCDRs (e.g., VHCDR1, VHCDR2 and/or VHCDR3) of (ii) those sequences (sequence of definition in (i)).In embodiments, antibody molecule, fragment or derivatives thereof comprise VHCDR1, VHCDR and VHCDR3 of SEQ ID NO:1 or VHCDR1, VHCDR and VHCDR3 of SEQ ID NO:3.In embodiments, antibody molecule, fragment or derivatives thereof comprise VHCDR1, VHCDR2 and VHCDR3 of SEQ ID NO:1 and are no more than 1 with the difference of VHCDR1, VHCDR2 and VHCDR3 of SEQ ID NO:1 on the whole, are no more than 2, are no more than 3, are no more than 4 or be no more than 5 amino acid.In embodiments, antibody molecule, fragment or derivatives thereof comprise VHCDR1, VHCDR2 and VHCDR3 of SEQ ID NO:3 and are no more than 1 with the difference of VHCDR1, VHCDR2 and VHCDR3 of SEQ ID NO:3 on the whole, are no more than 2, are no more than 3, are no more than 4 or be no more than 5 amino acid.

Antibody molecule, fragment or derivatives thereof optionally can also comprise the VL domain sequence that (i) and VL structural domain described in the invention (the VL structural domain of such as SEQ ID NO:2) have at least 60,70,80,85,90,95,98 or 99% amino acid sequence identity, or one group of VLCDRs (e.g., VLCDR1, VLCDR2 and/or VLCDR3) of (ii) those sequences (sequence of definition in (i)).In embodiments, antibody molecule, fragment or derivatives thereof comprise VLCDR1, VLCDR2 and VLCDR3 of SEQ ID NO:2.In embodiments, antibody molecule, fragment or derivative comprise VLCDR1, VLCDR2 and VLCDR3 and are no more than 1 with the difference of VLCDR1, VLCDR2 and VLCDR3 of SEQ ID NO:3 on the whole, are no more than 2, are no more than 3, are no more than 4 or be no more than 5 amino acid.The algorithm that can be used in calculating two aminoacid sequence percentage identities comprises such as BLAST, FASTA, or Smith-Waterman algorithm, as adopted default parameter.

IL-6a described in the invention can comprise antibody constant region or its part, as people's antibody constant region or its part.Such as, VL structural domain can comprise the light chain of antibody constant domain of people CK or CL chain in the attachment of its C-end.Similar, IL-6a based on VH structural domain can at all or part of (as the CH1 structural domain) of its C-end attachment heavy chain immunoglobulin, this heavy chain comes from any isotype antibody, as IgG, IgA, IgE and IgM and any isotype subclass, particularly IgG1, IgG2, IgG3 and IgG4.

In some cases, antibody of the present invention is used further methods known in the art and modifies the sequence with preparation with specific allotype (allotype), such as, in a kind of crowd having specific region origin prevailing allotype.In some cases, human heavy chain constant domain is modified to reach this object.

IL-6a can be antibody molecule, its binding fragment or variant, and it has the one or more CDRs in antibody framework, such as one group of CDRs.Such as, one or more CDRs or group CDRs of antibody (antibody such as described in the invention or its fragment or derivative) can be transplanted in framework (as people's framework) to provide antibody molecule.Framework region can be derived from human germline gene's sequence, can be maybe non-germline source.

VH and/or VL Framework residues can be modified also in the present invention as an example as discussed above, as used mutation site-specific.

Can methods known in the art and parameter be used to carry out amino acid change in the one or more framework region of antibody I L-6a (being defined as in the present invention " with reference to IL-6 antibody ") of site II being derived from target IL-6 and/or one or more CDRs.Also comprise produced IL-6 antagonist in the present invention, it remains with the combination of the site II of IL-6 (the site II as people JL-6) and usually has at least identical combination or the avidity of raising with compared with IL-6 antibody.In some cases, in order to improve the parameter of such as stability, can introduce and cause with the change of the binding affinity of reduction compared with IL-6a (as reference antibody) to prepare useful IL-6a.In some embodiments, such as in some cases, wherein said reference relates to FcRn and combines or pharmacokinetics (PK) parameter, as the transformation period in vitreum or whole body transformation period (as, at blood, blood plasma, serum, lymph, liver, kidney, its hetero-organization or body fluid), reference antibody can right and wrong specifically in conjunction with the antibody of IL-6.

Change in the aminoacid sequence of IL-6a polypeptide can comprise and replace one or more amino-acid residue with non-natural generation or non-standard amino acid, be that non-natural occurs or non-standard form by one or more Modification of amino acid residues, or insert the generation of one or more non-natural or non-standard amino acid in the sequence.The quantity changed in sequence of the present invention and the example of position describe in other places of the present invention.The amino acid of natural generation comprises 20 kinds of " standard " L-amino acid, and it is confirmed as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R by their Standard single-letter, H, D, E.Non-natural generation amino acid comprises any other residue that can infiltrate polypeptide backbone or be derived from existing Modification of amino acid residues.Non-standard amino acid can be the amino acid that natural generation or non-natural occur.The non-standard amino acid of several natural generations known in the art, as 4-Hydroxyproline, 5-hydroxylysine, 3-Methyl histidine and N-acetylserine.Those only will can be positioned at the N-end of aminoacid sequence at the amino-acid residue of N-alpha position derivatize.Amino acid is typically L-amino acid.In some cases, amino acid is D-amino acid.Therefore transform to comprise and by amino acid modified for L-be, or replaced with D-amino acid.Amino acid whosely to methylate, acetylize and/or phosphorylation form be known, and the amino acid in the present invention can carry out this kind modifies.

Aminoacid sequence in antibody domain of the present invention and binding members can comprise the non-natural or non-standard amino acid that the present invention discusses.Method as known in the art can be used, such as, modify or amino acid whose replacement after the synthesis of molecule or synthesis, non-standard amino acid (as D-amino acid) is infiltrated aminoacid sequence.In some cases, D-amino acid is used to the PK increasing IL-6a.

The mode that VH and/or VL nucleotide sequence by the one or more selection of random mutation produces sudden change in whole variable domains produces new VH or the VL district that the present invention carries CDR derived sequences.Such as can use fallibility PCR (Chao etc., Nature Protocols, 1:755-768 (2006)).In some embodiments, in whole variable region or one group of CDRs, one or two amino acid replacement is carried out.Other methods as known in the art can be used to produce sudden change, such as general rite-directed mutagenesis in one or more CDRs.

A method for generation of antibody I L-6a changes VH structural domain by adding, lacking, replace or insert one or more amino acid, as those are described in the structural domain of SEQ ID NO:1 and SEQ ID NO:3.The structural domain changed can with the VL domain combination being such as described in SEQ ID NO:2, described VL structural domain also can use method as known in the art as described in the present invention mode be changed.Test these altered molecules in conjunction with the ability of the site II of IL-6 and optionally test the avidity of other desired characteristic as increase compared with reference molecule.In some cases, variant VH or VL structural domain can have 1,2,3,4 or 5 such change (as 1,2,3,4 or 5 amino acid is replaced).

IL-6a of the present invention can be the fragment of the antibody of site II in conjunction with IL-6, as long as described fragment comprises antigen binding site, if the antigen binding site of site II in conjunction with IL-6.Antibody fragment of the present invention generally obtains, as comprised the antibody molecule of SEQ ID NO:1 and SEQ ID NO:2 or SEQ ID NO:3 and SEQ ID NO:2. from reference to (parent) antibody molecule.The mode of method as known in the art as the chemical chop (chemical reduction as disulphide bridges) of recombinant DNA, enzyme cutting (as used stomach en-or papoid), antibody can be used to produce antibody fragment.The antibody fragment comprising antibody antigen-binding site include, but are not limited to molecule as Fab, Fab ', Fab '-SH, scFv, Fv, dAb, Fd and the stable variable region (dsFv) of disulfide linkage.Other antibody multiple comprising one or more antibody antigen-binding site can be transformed, comprise such as F (ab ') 2, F (ab) 3, double antibody, three chain antibodies, four chain antibodies and mini antibody.The example of antibody molecule and their structure and using method is described in Holliger and Hudson, 2005, Nat Biotechnol 23:1126-1136.The example of the indefiniteness of binding fragment is the Fab fragment be made up of VL, VH, constant light structural domain (CL) and constant heavy structural domain 1 (CH1) structural domain; The Fd fragment be made up of VH and CH1 structural domain; The Fv fragment be made up of VL and the VH structural domain of single-chain antibody (single antibody); The dAb fragment be made up of VH or VL structural domain; The CDR district be separated; F (ab ') 2 fragment, the bivalent fragment of Fab fragments connected containing two; Single Chain Fv Molecule A (scFv), wherein VH structural domain and VL structural domain are connected by peptide connexon (linker) thus combine two structural domains to form antigen binding site; The double antibody of Bispecific single chain Fv dimer (as being disclosed in WO 1993/011161) and the multivalence using gene fusion (as being disclosed in WO94/13804) to build or bispecific antibody fragments.Stable Fv, scFv or double antibody is come by infiltrating the disulphide bridges connecting VH and VL structural domain.Can also use and comprise scFv and be connected to the mini antibody of CH3 structural domain as IL-6a.Other can be used as the fragment of the antibody of IL-6a and derivative comprises Fab ', it is different from Fab fragment by adding several amino-acid residue (comprising one or more halfcystines of antibody hinge region) at the C-terminal of heavy chain CH1 structural domain, and comprises the Fab '-SH of cysteine molecule with a free sulfhydryl groups of constant region.

In some cases, IL-6a is antibody fragment, and it has been modified by sulphation to strengthen or introduce the characteristic needed, as PEGization or to increase transformation period or integration.

DAb (domain antibodies) is the minor comonomer Fab (variable region of heavy chain of antibody or light chain of antibody.VH dAbs is natural to betide in camellid (such as camel and yamma) and by with target antigen immunity camel class animal, is separated antigen-specific b cells and direct mode of cloning dAb gene from single B cell produces.IL-6a of the present invention can be the dAb comprising VH or VL structural domain in the present invention substantially, or comprises VH or the VL structural domain of one group of CDRs in the present invention substantially.

Antibody of the present invention comprises bi-specific antibody, and the structural domain that two of this antibody are different is incorporated in same molecular.IL-6a can be infiltrated as a part for bi-specific antibody, described antibody can use means known in the art to prepare, such as chemical preparation or prepare from hybridoma.This molecule can be the bispecific antibody fragment of the type discussed above.The nonrestrictive example of method producing bi-specific antibody is BiTETM technology, can use the binding domains with not homospecific two antibody and directly connected by short flexible peptide in this technology.Two Antibody Combination are become a short single chain polypeptide by this.Can not use Fc district and only use variable domains to build double antibody and scFv, this alleviates anti-idiotype reaction potentially.Bi-specific antibody can be configured to whole IgG, is configured to dual specific Fab ' 2, is configured to Fab ' PEG, is configured to double antibody or is configured to other dual specifics scFv.In addition, two kinds of bi-specific antibodies can utilize ordinary method as known in the art to carry out connecting to form tetravalent antibody.

Dual specific double antibody, relative with dual specific complete antibody, being useful, is because they can be fabricated and express in intestinal bacteria to a certain extent.Phage display (WO 1994/13804) can be used from library easily to select the double antibody (and other polypeptide many, as antibody fragment) of suitable binding specificity.If keep an arm of double antibody constant, such as, directly for the specificity of the site II of IL-6, then can produce the variable library of other arms, thus select the antibody of appropriate specificity.

Dual specific whole antibody is described in the improvement project method preparation of WO 1996/27011, WO 1998/50431 and WO 2006/028936 by other.

In some cases, IL-6a of the present invention comprises antigen binding site in non antibody molecule, such as, by merging one or more CDRs, as group CDRs of in non-antibody protein skeleton, will discuss further hereinafter.In some cases, CDRs is integrated in the skeleton of non-antibody.By providing the binding site of the site II of IL-6 at non-antibody protein skeleton (as fibronectin or cytochrome B) upper arrangement CDRs, or given the binding specificity of the site II to IL-6 by amino-acid residue that is random or mutain skeleton inner ring (loop).Skeleton for the new binding site of through engineering approaches in albumen is that prior art is known.Peptide backbone such as antibody analog is disclosed at WO200034784, which depict the albumen (antibody analog) comprising the fibronectin type III domain with at least one randomized ring.The structural domain member of any immunoglobulin superfamily can provide and be suitable for being implanted into one or more CDRs, as the skeleton of one group of HCDRs.Skeleton can be people or non-human protein.The advantage of non-antibody protein skeleton is that it can provide antigen binding site in molecule of the skeleton, and described molecule of the skeleton is at least less than some antibody molecules and/or more easily prepare.Undersized binding members can bring useful physiological property, as the target position entering the ability of cell, degree of depth penetrate tissue or arrive in other structures, or in conjunction with the ability in target antigen albumen cave.Typically have the protein of stable main chain and one or more variable loop, in variable loop, the aminoacid sequence of a ring or multiple ring is had in conjunction with the specific antigen binding site of target antigen to produce by special or random mutation.This albumen comprises from the IgG binding domains of streptococcus aureus (S.aureus) a-protein, Transferrins,iron complexes, tetranectin (tetranectin), fibronectin (the type III structural domain as the 10th fibronectin) and NGAL (lipocalins) and γ-crystal and other Affilin ^tMskeleton (Scil Proteins, Halle, Germany).The example of additive method comprises the microbody of synthesis, and it is based on cyclic peptide (cyclotides)--there is the small protein of intramolecular disulfide bond (as Versabodies ^tM, Amunix Inc., Mountain View, CA) and ankyrin repeat protein (DARPins, as from Molecular Partners AG, Zurich-Schlieren, Switzerland).These albumen also comprise little, the protein structure domain of transformation, e.g., and immune structure territory (see such as, U.S. Patent Publication the 2003/082630th and No. 2003/157561).At least one complementary determining region (CDR) of antibody is contained in immune structure territory.

IL-6a can comprise extra amino acid, as the amino acid of other functional characters of molecule except the ability of conjugated antigen as described in giving.

In some cases, IL-6a carries a detectable mark, or puts together (e.g., by peptide bond or connexon) with toxin or targeting moiety or enzyme.Such as, IL-6a can comprise a catalytic site (as in enzyme domains) and an antigen binding site (binding site as the site II for IL-6), like this, antigen binding site be combined with antigen and therefore target catalytic site to IL-6 or IL-6/IL-6R mixture.Described catalytic site can, in some cases, suppress further the biological function of IL-6, as by cutting IL-6, IL-6R, or other molecules relevant with IL-6a/IL-6 mixture.

In certain aspects, to the present invention includes compared with reference antibody adorned antibody I L-6a and to change, such as, increase, reduce or eliminate the biological effect of IL-6a.In one embodiment, Fc district is modified or is replaced parent Fc structural domain with the pharmacokinetics of the IL-6a changing (compared with the parent of unmodified) and modify with the Fc structural domain modified.In some embodiments, IL-6a is through transforming to have IgG2 framework.In other embodiments, IL-6a in IgG1 or IgG2 framework has the Fc of modification, compared with parent or other references IL-6a, described IL-6a add binding affinity when pH6.0 and at pH7.0 time substantially do not change binding affinity, or Fc structural domain is modified and with parent or other are with reference to compared with IL-6a, the IL-6a of modification has the transformation period of increase.

In some embodiments, antibody I L-6a is modified to increase complement combination and CDC.In other respects, antibody I L-6a is modified to increase the ability of the cytotoxicity (ADCC) of (compared with reference antibody) antibody activation effect cell and participation antibody-dependant.In some cases, disclosed in the present invention antibody can be modified to strengthen its activation effect cell and participates in the ability of antibody-mediated cytotoxicity (ADCC) and increase the ability of the combination of its complement and participation CDC (CDC).

In some embodiments, antibody disclosed in this invention is modified to reduce its complement-fixing and is participated in the ability of CDC (CDC).In another embodiment, antibody is modified to reduce the ability of the cytotoxicity (ADCC) of its antibody activation effect cell and participation antibody-dependant.In yet, antibody disclosed in this invention can be modified to reduce its activation effect cell and participates in the ability of antibody-mediated cytotoxicity (ADCC) and reduce the ability of the combination of its complement and participation CDC (CDC).

Avoid sending IL-6a dosage continually normally favourable, such as, when injected delivery enters eyes.In order to promote this feature, in some embodiments, IL-6a disclosed in this invention is at least 4 days, such as at least 7 days, at least 9 days, at least 11 days or at least 14 days sending site such as the Vitrea transformation period.In some embodiments, the mean half-life of IL-6a is at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 25 days, 30 days, 40 days, 50 days or 60 days.The method increasing antibody half life is as known in the art, such as, be described in United States Patent (USP) the 6th, 277, No. 375 and No. 1998/23289, International Publication WO and No. 1997/3461, WO.In some embodiments, IL-6a in targeted delivery site as the Vitrea transformation period is greater than the whole body transformation period, the transformation period as in blood, blood plasma, lymph, liver, kidney or its hetero-organization or body fluid).

In other embodiments, the invention provides a kind of manufacture comprising container.Described container comprises the composition containing IL-6a disclosed in this invention, and instruction said composition can be used for package insert or the label for the treatment of IL-6 associated conditions.Usually, said composition is the composition comprising IL-6a and pharmaceutically acceptable vehicle.

In some cases, the present invention is the test kit of the explanation of experimenter's applying said compositions for the treatment of to needs containing the composition and instruction user that comprise IL-6a disclosed in this invention.

In the embodiment needing large IL-6a, such as strengthen IL-6a be positioned at or sending the reservation of location proximate, increase size but can combine with IL-6a the part that the function (binding affinity as to IL-6 or IL-6/IL-6R mixture) of IL-6a has a negative impact indistinctively.Such as can contain Fab and Fc single polypeptide partly to express by genetically engineered Fab.

In the embodiment of IL-6a needing relative small size, antibody or the fragment of IL-6 can be used, such as scFv or Fab fragment.The size of IgG antibody is about 150kD, and Fab is about 50kD, and scFv is about 25kD.In some embodiments, the size of IL-6a described in the invention is less than about 50kD.This antagonist can be, such as, is less than or equal to 50kD and is greater than 10kD, is less than or equal to 50kD and is greater than 20kD, is less than or equal to 50kD and is greater than 25kD.

In some cases, improve the stability of IL-6 antagonist (as there is antibody or other inhibitor of disulfide linkage) by manufacturing variant, wherein more stable than parent molecules of one or more disulphide bridgeses of this variant.

Other advantages of some IL-6a molecule described in the invention can be the operabilities of effective molecule, and described effective molecule has suitable mode of sending, sends the size of site or active patterns.Such as, the IL-6a of Fab form can be used for topical application.The method transforming this molecule is described in the present invention and be known in the art.

Indication/IL-6 relative disease

The disease of available IL-6a of the present invention treatment comprises these diseases, and the IL-6 raised in these diseases is relevant to morbid state or as the prerequisite of morbid state.Such disease comprises the disease pathology causing blood vessel generation and inflammation driven by IL-6.This comprises the IL-6 of rising compared with normal level, such as, in vitreum IL-6 raise disease (as diabetic macular edema, diabetic retinopathy and uveitis) or ocular tissue in IL-6 raise disease.The example of some illness in eye includes but not limited to dry eye syndrome, uveitis, age-related macular degeneration (AMD), proliferative diabetic retinopathy PDR (PDR), diabetic macular edema (DME), retinal vein occlusion (RVO), optic neuromyelitis (NMO).Other ophthalmic treatable comprises those and causes illness in eye by wound, as corneal transplantation, corneal abrasion, or other such physical injuries that eyes are caused.Therefore, the present invention includes the method using IL-6a treatment described in the invention to suffer from the experimenter of IL-6 relative disease.In some embodiments, the treatment of experimenter also comprises determines whether experimenter suffers from IL-6 relative disease, and optionally, whether patient resists other non-IL-6 suppression therapies, as steroid or anti-vegf treatment.

Some problem based on Antybody therapy is antibody at specific position as eyes, such as, in vitreum, be effective, but systemic administration can cause side effect.It is the therapeutical agent of example that a solution is to provide a kind of molecule as described in the present invention, and relative to systemic delivery, this therapeutical agent can local delivery.Because the therapeutical agent of some local deliveries (as being delivered in vitreum) there will be systemic delivery to a certain extent, the molecule therefore designed having relatively fast whole body and remove (systemic turnover) is favourable.Applicant has devised an example of this molecule; A kind of being designed to such as can the IL-6a antibody removed of quick systemic compared with parent molecules or reference antibody.This is combined by the FcRn of decorating molecule to realize with the circulation of the IL-6a reducing FcRn mediation.The further advantage of this design (as reduced FcRn circulation) is can by reducing along with IVT administration of antibodies improves its stop vitreum from Vitrea outflow.Therefore, in some embodiments, the IL-6a with low FcRn combination can reduce administration frequency.

Diabetic macular edema (DME).Diabetic macular edema (DME) participates in obstruction and the seepage of retinal vessel, causes visual deterioration and potential blind.Topical application steroid or VEGF antibody are comprised to the standard treatments of DME.But a lot of patient is refractories to these therapies.The pathogeny of diabetic macular edema relates to blood vessel generation, inflammation and oxidative stress.IL-6 is induced by anoxic and hyperglycemia and can be increased vascular inflammation, vascular permeability and pathologic vessels and occurs.IL-6 directly can induce the expression of VEGF and can promote choroidal neovascularization in animal model.In DME patient, the severity positive correlation of the IL-6 level of eyes and macular thickness and disease.IL-6 level is reported in and raises in the patient of anti-vegf therapy failure and reduce in the patient of anti-vegf response.Therefore, use IL-6a described in the invention and anti-vegf therapeutic combination using treat diabetic subject or as anti-vegf treat to substitute (comprise those and anti-vegf is treated to the patient do not responded) be useful.Treating macular edema with IL-6a also suppresses any one mechanism to suppress the demand of illness to improve its security by removing completely, thus remains some physiological actions needed of each cytokine.Therefore, local I L-6a treatment and VEGF suppress combined and can reduce administration frequency and alleviate the side effect for the treatment of.

In DME, experimenter's positive correlation of vitreum IL-6 level and disease severity and VEGF refractory.Therefore the IL-6a described in the present invention can be used for treating the DME experimenter to Steroid treatment, anti-vegf therapy or both refractories.In some cases, IL-6a and anti-vegf therapy or Steroid treatment combinationally use, as treatment DME.

IL-6a described in the present invention also can be used for treatment illness such as cancer, as prostate cancer (prostate cancer), leukemia (leukemia), multiple myeloma (multiple myeloma), inflammatory disease (as chronic inflammatory diseases proliferative disease (chronic inflammatory proliferative diseases)) and autoimmune disorder (autoimmune disease), as rheumatoid arthritis (rheumatoid arthritis), castleman's disease (Castleman ' s disease) (huge or blood vessel folliculus lymph node hyperplasia (giant or angiofollicular lymph node hyperplasia), lymph progonoma (lymphoid hamartoma), Angiofollicular lymph node hyperplasia (angiofollicular lymph node hyperplasia)), juvenile idiopathic arthritis (juvenile idiopathic arthritis) (comprising multi-joint juvenile idiopathic arthritis (polyarticular juvenile idiopathic arthritis) and systemic juvenile idiopathic sacroiliitis (systemic juvenile idiopathic arthritis)), Still disease (Still ' s disease) (comprising juvenile idiopathic arthritis (juvenile idiopathic arthritis) and adult Still disease (adult onset Still ' s disease)), adult Still disease (adult onset Still ' s disease), amyloid A amyloidosis (amyloid A amyloidosis), polymyalgia rheumatica (polymyalgia rheumatica), palliative seronegativity symmetry synovitis companion's pitting edema (remitting seronegative symmetrical synovitis with pitting edema), arthritis vertebralis (spondyloarthritides), behcets disease ( disease) (treatment for the treatment of eye manifestation (ocular manifestations) is comprised), atherosclerosis (atherosclerosis), psoriatic (psoriasis), systemic lupus erythematous (systemic lupus erythematosis), polymyositis (polymyositis) (inflammatory myositis inflammatory myopathy), relapsing polychondritis (relapsing polychondritis), Acquired hemophilia A (acquired hemophilia A), multiple sclerosis (multiple sclerosis), struvite anaemia (anemia of inflammation), with Crohn's disease (Crohn ' s disease).

IL-6 antagonist also can be used for treating some sacred disease, such as dysthymia disorders and Alzheimer.

IL-6a described in available the present invention treats other diseases, include but not limited to Systemic sclerosis (systemic sclerosis), multiple takayasu arteritis (Takayasu arteritis), giant cell arteritis (giant cell arteritis), graft versus host disease (GVH disease) (graft versus host disease), with Tumor Necrosis Factor Receptors associated period syndromes (TNF-receptor-associated periodic syndrome TRAPS).

Dosage

IL-6 antibody or its fragment can be administered to experimenter (as patient), described experimenter expresses, such as abnormal high-caliber IL-6.Antibody or its fragment can applied onces, or can repeatedly use.Can administration of antibodies, such as, from every day three times to every six months once or more of a specified duration.Can use according to plan as every day three times, twice daily, once a day, every two days once, every three days once, once in a week, once every two weeks, monthly, every two months once, every three months once with every six months once.Antibody or fragment can continue to use as implantable slowly-releasing glue skirt or by the cell producing antibody or its fragment via mini-pump or other approach.Antibody or its fragment can via in mucous membrane, oral cavity, nose, suction, intravenously, subcutaneous, intramuscular, parenteral, intraocular or intratumoral route administration.Antibody or its fragment can be used once, at least twice or use within the time period that at least illness is treated, alleviates or cures.General by administration of antibodies or its fragment, as long as illness exists.Antibody or its fragment, generally it can be used as a part for pharmaceutical composition described in the invention to use.The dosage of antibody generally will 0.1 to 100mg/kg, 0.5 to 50mg/kg, 1 to 20mg/kg and 1 in the scope of 10mg/kg.The serum-concentration of antibody or its fragment is measured by any suitable method.One of some compound described in the invention is characterised in that their need administration relatively infrequently, such as once in a week, twice weekly, on every Wendesdays secondary, every surrounding once, once every two weeks, every 8 weeks once, every 12 weeks once, every 16 weeks once, every 32 weeks once, monthly, every two months once, every three months once or every six months once.In some cases, on an as-needed basis, using of (such as by the state of experimenter) compound is determined.One of IL-6 antagonist of permission described in the invention relatively infrequently administration is characterised in that combined for the ability of efficient (at least partly by once be attached to IL-6, have slow dissociation rate and realize) and the compound of sending rather high concentration.

In some cases, IL-6a uses as monotherapy.In other embodiments, IL-6a and methotrexate or Other diseases improve arthritis medicine and use simultaneously.

The generation of antibody

Means known in the art can be used as monoclonal antibody methodology (e.g., see Kohler and Milstein (1975) Nature 256:495) production antibody I L-6a or derivatives thereof or fragment.The technology of other manufacture monoclonal antibodies also can be adopted as the virus of bone-marrow-derived lymphocyte or neoplastic transformation.

Can be fitted together to or humanized antibody based on the mouse monoclonal antibody preparation using means known in the art to prepare.The DNA of encoding heavy chain and light chain immunoglobulins can obtain from interested Mouse Hybridoma Cells, and described DNA can use standard molecular biological technique to transform with the immunoglobulin sequences comprising non-mouse (as people).Such as, in order to prepare chimeric antibody, technology known in the art (see such as No. the 4th, 816,567, United States Patent (USP)) can be used to be connected with human constant region mouse variable region.In order to manufacture humanized antibody, technology known in the art can be used (see such as No. the 5th, 225,539, United States Patent (USP) and the 5th, 530,101; 5,585,089; 5,693, No. 762; With the 6th, 180, No. 370) mouse CDR district is inserted people's framework.

In embodiments, IL-6a (as anti-IL-6 antibodies or derivatives thereof or fragment) described in the invention can specifically in conjunction with people IL-6.In embodiments, IL-6a can specifically in conjunction with the site II (the site II as people IL-6) of IL-6.

In some embodiments, IL-6a antibody is human monoclonal antibodies.This antibody can use and comprise groups of people's immunity system but not the transgenosis of mouse system or transchromosomic mice produce.These transgenosiss and transchromosomic mice comprise in the present invention and are referred to as HuMAb and KM mouse, and be referred to as in the present invention " people Ig mouse " (see U.S. Patent number 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299 and 5,770, No. 429; U.S. Patent number 5,545,807; PCT publication number WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962; With No. 01/14424, PCT publication number WO).

In other respects, can be used in the mouse of carrier's immunoglobulin sequences in transgenosis or transfection chromosome, the mouse as carrier's heavy chain transgene and people's light chain transfection chromosome produces people's anti-IL-6 antibodies.This kind of mouse is described in detail in PCT publication number WO 02/43478.

Other transgenic animal systems expressing human immunoglobulin gene are that this area is obtainable, and can be used to production antibody I L-6a.Such as can use and be called Xenomouse ^tMthe alternative transgenic system of (Abgenix, Inc.); This type of mouse is described in such as U.S. Patent number 5,939,598; 6,075,181; 6,114,598; 6,150,584; With 6,162, in 963.In addition, the trans-chromosome animal system expressing human immunoglobulin gene is that this area is obtainable, and can be used for production antibody I L-6a.The such as mouse of carrier's heavy chain transchromosome and people's light chain transfection chromosome is described in Tomizuka etc. (2000, Proc Natl Acad Sci USA 97:722-727).SCID mouse can also be used to prepare human monoclonal antibodies, wherein in this mouse, rebuild people's immunocyte thus people's antibody response can have been produced by immunization.This type of mouse is described in such as U.S. Patent number 5,476,996 and 5,698, in 767.

Phage display library

In some cases, antibody I L-6a antibody or derivatives thereof or fragment be using phage synthesis people antibody library, screen described library with IL-6 (such as people IL-6) or its fragment, the phage of separation and combination IL-6 and obtaining the method for antibody from described phage produces.

Separating recombinant human antibody IL-6a can also be carried out by screening restructuring combinatorial antibody library.In general, described library is scFv phage library, and this library is that employment VL produces with VH cDNAs (preparing from the mRNA of B cell from being separated).It is known in the art for preparing and screening this kind of library.Test kit for generation of phage display library be commercially available (as the recombinant phages antibody system of Pharmacia company, catalog number (Cat.No.) 27-9400-01; With Stratagene SurfZApTM phage display test kit, catalog number (Cat.No.) 240612).Other can be used for producing and the method for screening antibodies phage library and reagent be known in the art (see, such as U.S. Patent number 5,223,409; PCT publication number WO 92/18619, WO 91/17271, WO 92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs etc., Bio/Technology 9:1370-1372 (1991); Hay etc., Hum Antibod Hybridomas 3:81-85 (1992); Huse etc., Science 246:1275-1281 (1989); McCaffert etc., Nature 348:552-554 (1990); Griffiths etc., EMBO J 12:725-734 (1993); Hawkins et al., J Mol Biol 226:889-896 (1992); Clackson etc., Nature 352:624-628 (1991); Gram etc., Proc Natl Acad Sci USA 89:3576-3580 (1992); Garrad etc., Bio/Technology 9:1373-1377 (1991); Hoogenboom etc., Nuc Acid Res 19:4133-4137 (1991); With Barbas etc., Proc Natl Acad Sci USA 88:7978-7982 (1991), be all incorporated to as a reference herein.

Being separated at one and producing has in the example of IL-6 antibody of required feature, first employment IL-6 antibody, use epi-position blotting to select heavy chain and the sequence of light chain of similar IL-6 binding ability, described method is described in PCT publication number WO 93/06213, incorporated herein by reference.The antibody library used in this method is generally scFv library, and the preparation in this library and screening are described in PCT publication number WO 92/01047; McCafferty etc., Nature 348:552-554 (1990); And Griffiths etc., EMBO J 12:725-734 (1993), be all incorporated to herein as a reference.

Once have selected initial people VL and VH structural domain, just carry out " mix and mate " experiment, the IL-6 binding ability of VL and the VH fragment of the different right initial selected of screening is to select preferred VL/VH to composition in this experiment.In order to select the feature required for IL-6a, VL and/or VH fragment that can be right selected by random mutation.This external Affinity maturation is such as by using the mode of VH and the VL structural domain that to increase with of selected VH and VL structural domain or both PCR primer of CDR complementation to realize, wherein said primer is included in the random combine of four nucleotide bases in some site, thus the PCR primer produced coding introduces the fragment of VH and/or VL of random mutation.VH and the VL fragment of this kind of random mutation can be used to the binding fragment screening IL-6, the binding fragment of the site II of such as IL-6.

After screening from the immunoglobulin display library of recombinating and separation antibody IL-6a, the nucleic acid of the antibody of codes selection can be reclaimed from demonstration package (display package) (such as from phage genome), and by recombinant DNA technology known in the art, its subclone is entered other expression vectors.Can operate further to produce antibody fragment to this antibody-like, as the antibody fragment that those are recorded in the present invention.

Pharmacokinetics (PK)

Means known in the art can be used to carry out PK test.Need to use animal to carry out the obstacle of measurement example as measured PK and be that the IL-6 of the mankind and those IL-6 being generally used for some animals of this class testing has the homology being less than 50%.A kind of method of therefore testing PK uses the transgenic mice of expressing people IL-6.In some embodiments, non-human primate is used to measure PK.

In some embodiments, anti-IL-6 antibodies be sudden change to change its PK, such as by change FcRn combine pH susceptibility and change its PK.The method obtaining this kind of sudden change is recorded in embodiment.Therefore, in some embodiments, compared with parent IL-6a or reference molecule, described IL-6a has the whole body PK (systemic PK) of change.In some cases, PK is immovable or improves in vitreum.In some embodiments, compared with parent IL-6a or reference molecule, the whole body PK (such as measuring in circulation fluid is as blood, blood plasma, lymph liquid, serum, liver, kidney, other tissue or other body fluid) that IL-6a has PK (transformation period as increased) that is identical or that increase and reduces in vitreum.

For the model of test I L-6 antagonist

Can in disease model the IL-6 associated delivery of test I L-6 antagonist, particularly test result for the treatment of and the limited side effect for IL-6 advantageous feature.Such as can in rat or mouse experiment Autoimmune uveitis model, to be used between the photosensory cell in complete Freund's adjuvant (CFA) acceptor retinoid associated proteins (IRBP) immunization to test uveitis (Caspi, Invest Ophthalmol Vis Sci 52:1873; Agarwal etc., 900:443-69,2012).Other models comprise the uveitis of those Dendritic Cells Induced as known in the art, the adoptive transfer of the effector T cell of cultivation, the spontaneous EAU in IRBP TcR Tg mouse, the uveitis of endotaxin induction, autoimmune uveoretinitis (Haruta etc., Invest Ophthalmol Vis Sci 53:3264 (2011); Yoshimura etc., Rheumatology 48:347-354 (2009)).Other model systems that can be used in measuring IL-6a disease effects have such as, choroidal neovascularization (CNV) model (Izumi-Nagai etc., Am J Pathol 170:6 (2007); Krzystolik etc., Arch Ophthalmol 120:338 (2002)) and diabetes model be described in the (models of Animal Models Of Diabetic Complications Consortium (P01 DK57733), Update Report (September 2001-January 2004) such as Kern as those.Known in the art for the animal model of test I L-6a in rheumatoid arthritis, for example, see (Ann Rheum Dis 70:1357-62 (2011) such as Asquith etc. (Eur J Immunol 39:2040-4 (2009)) and Kollias.

Conjoint therapy

In some embodiments, by IL-6a and the second therapeutic entity (therapeutic entity) combined administration.Such as comprise VEGF inhibitor as the treatment plan of ranidizumab in use IL-6a.In some embodiments, in the treatment plan comprising PDGF inhibitor, use IL-6a, wherein said PDGF inhibitor is as anti-PDGF antibody or anti-pdgf receptor antibody (as imatinib).

Sending of IL-6 antagonist

IL-6 antagonist can local delivery, directly contact or contiguous target cell or the tissue carrying out IL-6 suppression.The nonrestrictive example of this delivering method comprises injection, transfusion or implants the material containing IL-6 antagonist.

In some embodiments, IL-6a composition is used as ophthalmic preparation.The method can comprise uses IL-6a composition and eye acceptable carrier.In some embodiments, ophthalmic preparation is liquid, semisolid, inset (insert), film, particulate or nano particle.

In some embodiments, IL-6a composition is formulated for topical, as ophthalmic administration.Described topical formulations can be liquid preparation or semisolid, and such as, topical formulations can comprise the aqueous solution, waterborne suspension, ointment or gel.Under the preparation of IL-6a of eye can be locally applied to the front portion of eyes, upper eyelid, on lower eyelid and in cul-de-sac.Usually, ophthalmic preparation is aseptic.IL-6a ophthalmic preparation can containing one or more drug excipients of preparation being suitable for ophthalmic preparation.The example of these vehicle is sanitas, buffer reagent, sequestrant, antioxidant and for regulating the salt of osmotic pressure.The ophthalmic preparation comprising ointment and suspension generally has the viscosity being suitable for selected administration route.In some embodiments, ophthalmic preparation has the viscosity of from about 1,000 to about 30,000 centipoise.

In some embodiments, described preparation is a kind of liquid preparation comprising polymkeric substance.Bioavailability, raising viscosity or reduction liquid preparation that this base polymer can be used for improving are discharged from eyes.Suitable polymkeric substance includes but not limited to, at those polymkeric substance that the people such as Wagh (Asian J Pharm, 2:12-17,2008) describe.In nonrestrictive example, polymkeric substance is that the sodium salt of Unidasa, chitin, cyclodextrin (as hydroxypropyl beta cyclodextrin), polygalacturonic acid, xyloglucan, xanthan gum, gelling gum, thiolated polymers, poly-(ortho ester) are (as Einmahl, Adv Drug Deliv Rev 53:45-73,2001) or tamarind seed polysaccharide (as, Ghelardi etc., Antimicrob Agents Chemother 48:3396-3401,2004).

In some embodiments, the IL-6a preparation comprised for ocular delivery can containing one or more tensio-active agent, auxiliary, adjuvant, antioxidant, tension regulator, sanitas is (as EDTA, BAK (benzalkonium chloride), Textone, Sodium peroxoborate, polyquaternium-1 (polyquaterium-1)), thickening material or viscosity modifier are (as carboxymethyl cellulose, Walocel MT 20.000PV, polyvinyl alcohol, polyoxyethylene glycol, ethylene glycol 400, propylene glycol Walocel MT 20.000PV, hydroxypropyl guar gum, hyaluronic acid and hydroxypropylcellulose) etc.Additive in preparation can include, but not limited to sodium-chlor, sodium bicarbonate, Sorbic Acid, methyl p-hydroxybenzoate, propylparaben, chlorhexidine (chlorhexidine), Viscotrol C and Sodium peroxoborate.

In some embodiments, pure water or deionized water can be used in composition.By adding the acid of any physiology and the acceptable pH regulator of eye, alkali or buffer reagent by pH regulator in the scope of about 5.0 to 8.5, as pH7.0, pH7.3, pH7.4 or pH7.5.Eye comprises acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, hydrochloric acid etc. with the example of acceptable acid, and the example of alkali comprises sodium hydroxide, sodium phosphate, Sodium Tetraborate, Trisodium Citrate, sodium acetate, Sodium.alpha.-hydroxypropionate, tromethane, trishydroxymethylaminomethane etc.The example of the salt and damping fluid that can be used for preparation comprises the mixture of Citrate trianion/dextrose, sodium bicarbonate, ammonium chloride and above-mentioned bronsted lowry acids and bases bronsted lowry.

In some embodiments, the osmotic pressure of ophthalmic composition can be that about 10 milli infiltration mole (milliosmolar) (mOsM) such as, to about 400mOsM, 200 to 400mOsM or 220 to 370mOsM.Usually, osmotic pressure can be regulated with on physiology with the acceptable salt of ophthalmology or vehicle.In some embodiments, preparation comprises sodium-chlor, and such as, the sodium-chlor concentration by weight be present in preparation is 0.01% to 1% or is 0.05% to 0.45% by weight, based on the gross weight of composition.Also can use by one or more equivalent by positively charged ion (as potassium, ammonium etc.) and negatively charged ion (as chlorion, citrate, Vitamin C acid group, borate, phosphate radical, bicarbonate radical, sulfate radical, thiosulfate anion, hydrosulfate, sodium pyrosulfate, ammonium sulfate etc.) salt that forms and sodium-chlor together or alternative sodium-chlor to realize the osmotic pressure in the scope of needs.In some embodiments, also can use sugar, as mannitol, dextrose, Sorbitol Powder, glucose etc., regulate osmotic pressure.

In some embodiments, described method comprises formation or provides and the bank of the medicament of eye exterior surface (depot of the agent).Bank refers to the supply source of reagent, and described bank can not be removed by tear or other eye purge mechanisms.This allows by single application, and medicament that is continuous, prolonged high concentrations is present in the fluid of outer ocular surfaces.In some embodiments, bank can keep nearly more than eight hours.In some embodiments, eye depot formulations includes but not limited to, aqueous polymer suspension, ointment and Solid inserts (solid inserts).

In some embodiments, semi-solid combination is a kind ofly administered to the liquid preparation at the moment increasing viscosity, described viscosity normally owing to there is polymkeric substance in liquid preparation, along with the change viscosity of temperature, pH or electrolyte concentration increases to some extent.Polymkeric substance can be such as, ethanoyl phthalate, cellulose (celluloseacetophthalate), polyacrylic acid, gelling gum; Unidasa; chitin, the segmented copolymer of alginate (as sodiun alginate) or oxyethane and propylene oxide (as bASF; Poloxamer).In some embodiments, polyacrylic acid be crosslinked vinylformic acid (e.g., ).In some embodiments, semi-solid combination contains the mixture of the segmented copolymer of carbomer and oxyethane and propylene oxide; The mixture of methylcellulose gum and Natvosol; Or the mixture of the segmented copolymer of polyoxyethylene glycol and oxyethane and propylene oxide.

In some embodiments, the ophthalmic preparation comprising IL-6a is ointment or gel.In some embodiments, ophthalmic preparation is oil-based delivery vehicle.Such as, preparation can comprise the oil or lanolin base carrier and vehicle that add IL-6a composition (as 0.1% to 2%).Common matrix (base) includes but not limited to mineral oil, vaseline and combination thereof.In some embodiments, ointment is applied to lower eyelid as band (ribbon).

In some embodiments, ophthalmic composition is eye inset (insert).Such as described eye inset be any biological inert, soft, bioerodable, viscoelastic (viscoelastic), be exposed to therapeutical agent after stablize sterilizing, to airborne transmission bacteriological infection resistance, bioerodable, biocompatible and/or viscoelastic (viscoelastic).In some embodiments, inset comprises the acceptable matrix of eye, such as, and polymeric matrix.Described matrix is generally a kind of polymkeric substance, and IL-6a composition is dispersed in Medium Culture or is attached to polymeric matrix.In some embodiments, medicament from matrix by dissolving or hydrolysable covalent bonds and slow releasing.In some embodiments, polymkeric substance is bioerodable (dissolving), and its dissolution rate can control the release rate being dispersed in polymeric matrix Chinese medicine.In another form, polymeric matrix is biodegradable polymkeric substance, and this polymkeric substance such as decomposes by being hydrolyzed, thus discharges and its combination or the medicament that is dispersed in wherein.In further embodiment, available other polymer coating encirclement matrix and medicament are with further Co ntrolled release.In some embodiments, inset comprises biodegradable polymkeric substance, as polycaprolactone (PCL), ethylene/vinyl acetate (EVA), polyalkyl alpha-cyanacrylate, urethane, nylon or poly DL-lactide-co-glycolide) (PLGA) or these multipolymer arbitrarily.In some cases, medicament is dispersed to substrate material or is dispersed in monomer composition, and described monomer composition is used for manufacturing substrate material before polymerization.In some embodiments, the amount of medicament is about 0.1 to about 50% or from about 2 to about 20%.Can use can the polymeric matrix of biodegradable or bioerodable, thus the inset that need not remove from eyes.Along with degraded or the dissolving of biodegradable or bioerodable polymkeric substance, described medicament is released.

In further embodiment, eye inset comprises a kind of polymkeric substance, include but not limited to, those are at Wagh, Deng, " Polymers used in ocular dosage form and drug delivery systems ", Asian J.Pharm., the polymkeric substance recorded in pages 12-17 (January 2008), is incorporated to herein by quoting in full.In some embodiments, inset comprises polymkeric substance, and described polymkeric substance is selected from polyvinylpyrrolidone (PVP), acrylate or methacrylate polymers or multipolymer (e.g., from Rohm's or Degussa polymeric families), Walocel MT 20.000PV, polyacrylic acid, poly-(amidoamines) branch-shape polymer, poly-(dimethyl siloxane), polyethylene oxide, poly-(lactide-co-glycolides), poly-(HEMA), polyvinyl alcohol) or poly-(propylene fumarate).In some embodiments, inset is the halfcystine-polyacrylic acid conjugate of 450kDa.

Inset can comprise core (core) containing IL-6a composition and outer tube (as being described in No. 20040009222nd, U.S. Patent Publication).In some cases, outer tube can be infiltrative to medicine, semi-permeable or impervious.In some embodiments, core comprises the polymeric matrix do not made a significant impact the release rate of IL-6a composition.In some cases, the polymeric matrix of outer tube, core or both are bioerodable.Coextrusion product (co-extruded product) may be partitioned into (segmented into) drug delivery device.In some embodiments, described device be do not wrap quilt to make respective end be open, or described device such as film (layer) wraps quilt, described film to IL-6a composition be permeable, be semi permeable or bioerodable to IL-6a composition.In some embodiments, IL-6a composition and at least one polymkeric substance mix in powder form.

In some embodiments, ophthalmic composition is eye film.The polymkeric substance being suitable for this type of film includes but not limited to that those are described in the polymkeric substance of (ditto) such as Wagh.In some embodiments, described film is soft contact lens (soft-contract lens), the glasses be such as made up of N, N-acrylamide and the multipolymer of methacrylic acid that is cross-linked with ethylene glycol dimethyl.

In some embodiments, IL-6a is arranged in the inset of tubulose, and can be segmentation (segmented).

In some embodiments, IL-6a composition is configured to treatment significant quantity, by polymeric matrix bag quilt or point in the polymer matrix, make IL-6a composition be granular or particle form.In some embodiments, when the medicine dissolution in particle to or in matrix, along matrix diffusion, and when being released in surrounding physiological fluid, IL-6a composition discharges from configuration thing.In some embodiments, rate of release is mainly subject to IL-6a composition from particle/particle dissolution to the restriction of the speed matrix; Along matrix diffusion and the step be distributed in surrounding liquid be not main rate of release limiting factor.In some embodiments, polymeric matrix is non-bioerodable, and it can biological corrosion in other embodiments.The exemplary abiotic corrodible polymeric matrix that formed can be urethane, polysiloxane, poly-(ethylene-co-vinyl acetate) (EVA), polyvinyl alcohol and derivative thereof and multipolymer.The exemplary abiotic corrodible polymeric matrix that formed can be polyanhydride, poly(lactic acid), polyglycolic acid, poe, Polyalkylcyanoacrylanano (polyalkylcyanoacrylate) and derivative thereof and multipolymer.

In some cases, in collagenic material, IL-6a composition is prepared.Such as, inset can be that solubility eye medicinal inset is (if be introduced in the oval polymeric film of conjunctival sac in order to drug delivery; Oval inset is as being made up of ethylene vinyl acetate (pilocarpine eye treatment system, is developed by Alza Corporation); the shaft-like inset be made up of Mierocrystalline cellulose; Novel ophthalmology delivery system (NODS), is made up of poly-(vinyl alcohol); Or those are described in the inset in Fabrizio (Adv Drug Deliv Rev 16:95-106,1998).In some cases, inset comprises collagen, gelatin or polymkeric substance, and wherein said polymkeric substance is selected from polycaprolactone (PCL), ethylene/vinyl acetate (EVA), cyanoacrylate, urethane, nylon, poly-(DL-lactide-co-glycolide (PLGA) or these multipolymer any.In some cases, under upper eyelid, inset is implanted.In some cases, inset is implanted in the back segment of eyes, suprachoroidal space or sclera.In some embodiments, in vitreum or under retina, inset is implanted.

In some embodiments, subretinal injection inset.The method used and the technology of preparing of inset are set forth in remington ' s:The Practice of Science of Pharmacy, 20th editionin (Lippincott Williams & Wilkins, 2006), by reference it is all incorporated to the present invention.

In other embodiments, the inset of the IL-6a composition comprised provides the sustained release of medicament to eye vitreous.As used in the present invention, " sustained release " refers to composition release medicine within the time extended in a controlled manner.In some embodiments, inset, with a kind of speed release medicine, makes in the dispose procedure of medicament, and in water, the concentration of the medicament of (aqueous agent) continues the concentration lower than vitreum Chinese medicine.In some embodiments, in water, the concentration of the medicament of (aqueous agent) is from about 0.002 μ g/mL to about 0.01 μ g/mL or from about 0.01 μ g/mL, arrives about 0.05 μ g/mL or arrives lower than about 0.05 μ g/mL.In some embodiments, with about 1 μ g/ days to about 50 μ g/ days, or from the speed release medicine of 1 μ g/ days to about 10 μ g/ days.In some embodiments, inset also comprises extra therapeutical agent as detailed above, and such as fluocinolone acetonide (as is present in ophthalmic insert in).

In some embodiments, ophthalmic composition comprises microballoon or nano particle.In some embodiments, described microballoon comprises gelatin.In some embodiments, microballoon is injected in the back segment of eyes, suprachoroidal space or sclera.In some embodiments, microballoon or nano particle comprise polymkeric substance, include but not limited to that those are at Wagh, wait the polymkeric substance described in (Asian J Pharm 2:12-17,2008).In some embodiments, polymkeric substance is chitin, poly carboxylic acid, as polyacrylic acid, albumin particle, hyaluronic acid ester, poly-methylene-succinic acid, poly-(butyl) cyanoacrylate, polycaprolactone, poly-(isobutyl-) caprolactone, poly-(lactic-co-glycolic acid) or poly-(lactic acid).In some embodiments, microballoon or nano particle comprise solid lipid particles.

In some embodiments, IL-6a composition comprises ion exchange resin.In some embodiments, ion exchange resin is the organic resin of inorganic zeolite resin or synthesis.In some embodiments, ion exchange resin includes but not limited to the middle resins (the same) described such as Wagh, by reference it is all incorporated to the present invention.In some embodiments, ion exchange resin is the polyacrylic acid of part neutralization.

IL-6a composition can be provided in waterborne polymeric suspension.In some embodiments, IL-6a composition or polymer suspension are suspended in aqueous medium (the aqueous medium as characteristic as described in having above).Polymerization flotation reagents example include, but not limited to dextran, polyoxyethylene glycol, polyvinylpyrrolidone, polysaccharide gel, cellulose polymer compound as Vltra tears and carboxylic polymkeric substance, as acrylic acid polymkeric substance or multipolymer, and other polymkeric substance negative catalyst (polymeric demulcents).In some embodiments, this polymkeric substance negative catalyst is water-swellable, insoluble polymer, particularly crosslinked carboxylic polymkeric substance.In some embodiments, in the gross weight of existing monomer for benchmark, this polymer suspension agent comprises at least about 90 % by weight to about 99.9%, or one or more carboxylic single ethylenically unsaturated monomers of about 95% to about 99.9%.In some embodiments, this carboxylic single ethylenically unsaturated monomer comprises vinylformic acid, methacrylic acid, ethylacrylic acid, methacrylic acid (β-crotonic acid), cis-tiglic acid (angelicic acid), trans-tiglic acid (tiglic acid), α-butyl β-crotonic acid, α-phenylacrylic acid, α-benzyl acrylic, α-cyclohexylacrylic, phenylacrylic acid (styracin), coumaric acid (o-hydroxy cinnamic acid) and umbellic acid (to hydroxyl coumaric acid).In some embodiments, this polymkeric substance is cross-linked by multi-group crosslink agent's (e.g., dual functional linking agent).In some embodiments, in the gross weight of existing monomer for benchmark, the content of this linking agent is about 0.01% to about 5%, or about 0.1% to about 5.0%, or about 0.2% to about 1%.In some embodiments, this linking agent is non-polyalkenyl polyether bi-functional linker monomer, such as DIETHYLENE GLYCOL, 2,3-dihydroxyl own-1,5 diene, 2,5-dimethyl-1,5-hexadienes, Vinylstyrene, N, N-diallyl acrylamide, N, N-diallyl Methacrylamide; Every a part contains the polyalkenyl polyether linking agent of two or more alkenyl ether groups, as the alkenyl ether groups containing end H2C=C group, by with haloolefin as the polyvalent alcohol etherificate containing at least four carbon atom and at least three hydroxyls is prepared by allyl bromide 98 etc., as polyallylsucrose, polyallyl pentaerythritol etc.; The diolefinic non-hydrophilic macromolecules cross-linking agent of molecular weight about 400 to about 8000, the insoluble diacrylate of such as glycol and polyvalent alcohol and many acrylate and methacrylic ester, the vulcabond acrylic or methacrylic acid hydroxy alkyl ester reaction product etc. of the prepolymer of the isocyanic ester terminal derived by polyester glycol, polyether glycol or silicone glycol and hydroxyalkyl methacrylate

In some embodiments, cross-linked polymer can be prepared by one or more carboxylic single ethylenically unsaturated monomers of the single ethylenically unsaturated monomer as unique existence and one or more linking agents.In some embodiments, polymkeric substance is wherein at the most about 40%, preferably one or more carboxylic single ethylenically unsaturated monomers of about 0% to about 20% are not contained carboxyl by one or more, only containing the polymkeric substance that substituent single ethylenically unsaturated monomer harmless with ophthalmology on physiology substitutes, described monomer comprises acrylate and methacrylic ester, such as methyl methacrylate, ethyl propenoate, butyl acrylate, 2-EHA, Octyl methacrylate, HEMA, vinylformic acid 3-hydroxypropyl acrylate etc., vinyl-acetic ester, NVPs etc. are (as Mueller etc., United States Patent (USP) the 4th, 548, No. 990).In some embodiments, polymkeric substance comprise polycarbophil (Noveon AA-1), with in some embodiments, the dry granularity that monomer suspension or letex polymerization to equivalent spherical diameter are no more than about 50 μm is prepared by using conventional radical polymerization catalyzer by this cross-linked polymer.In some embodiments, this average dry granularity is equivalent spherical diameter about 1 to about 30 μm, or about 3 about 20 μm.In some embodiments, polymer beads is by obtaining larger polymer beads mechanical mill.In other embodiments, this base polymer has about 250, and 000 to about 4,000,000, and 3,000,000,000 to 4,000, the molecular weight of 000,000.In other embodiments, the particle of cross-linked polymer is monodispersed, refers to that the size-grade distribution that they have should make to fall in μm band of main size-grade distribution at least about the particle of 80%, about 90% or about 95%.In other embodiments, single dispersing granularity refers to and is no more than about 20%, about 10%, or the granularity of about 5% particle is below 1 μm.In some embodiments, aqueous polymer suspension comprise about 0.05 to about 1%, about 0.1 to about 0.5% or about 0.1 to about 0.5% therapeutical agent and about 0.1 to about 10%, about 0.5 to about 6.5%, about 0.5 to about 2.0%, about 0.5% to about 1.2%, the polymer suspension agent of about 0.6 to about 0.9% or about 0.6 to about 0.8%.Although should be mentioned that single, should be appreciated that, when total amount is in specialized range, one or more polymer suspension agent can be used.In one embodiment, amount, the pH of insoluble lightly crosslinked polymer beads can be relative to each other with osmotic pressure, and relevant to degree of crosslinking, have about 500 to about 100,000 centipoise to provide, preferred about 1,000 to about 30,000 centipoise or about 1,000 to about 10, the composition of the viscosity of 000 centipoise, described viscosity uses the Brookfield numeral LVT viscometer that No. 25 mandrels and 13R small sample joint are housed to measure under 12rpm and room temperature (about 25 DEG C).In some embodiments, this viscosity is about 10 to about 400 centipoises, about 10 to about 200 centipoises or about 10 to about 25 centipoises.

In some embodiments, can prepared polymer waterborne suspension, make they keep in eye with deliver medicine to eyes before the identical or substantially the same viscosity of viscosity.In some embodiments, can be prepared them, its gelation when contacting with tear is strengthened.Such as, when pH is lower than about 6.7 contain or the preparation of other polypropylene-like acids polymers is when delivering medicine to eyes, this polymkeric substance may be swelling when contacting with tear, because it has higher pH (about 7).The enhancing of this gelation or gelation may cause carrying secretly of suspended particle, thus extends the residence time of composition in eyes.In some embodiments, along with suspended particle through time dissolve, this therapeutical agent discharges at leisure.In some embodiments, this transport way adds patient comfort, adds the duration of contact of therapeutical agent and ocular tissue, thus adds degree and the acting duration of preparation in eyes of drug absorption.The therapeutical agent contained in these delivery systems can depend on that such as medicine itself and profile thereof, the pH of degree that medicine loads and system and any drug conveying auxiliary agent that also may the exist speed as shown the factor of compatible ion exchange resin and so on eye discharges from gel.

In some embodiments, use gene delivery, as local heredity is sent, IL-6 antagonist is supplied to experimenter.This sending is sent by transient expression system, a kind of stable (as integrated) expression system is (as by Bluebird Bio (Cambridge, MA) the slow virus delivery system produced), or send in cell factory, as the cell factory that those are produced by Neurotech (Cumberland, Rhode Island).

All technical characteristics can all possible array mode of these features combine individually.

Equivalent

The present invention can embody with other other specific forms without prejudice to its spirit or principal character.Therefore aforementioned embodiments is considered to illustrative but not to invention of the present invention restriction in all respects.

Claims

1. the antibody be separated or its Fab, it can specifically in conjunction with the site II of people IL-6.

2. the monoclonal antibody be separated or its Fab, it comprises

VH CDR1 shown in a.SEQ ID NO:4, the VH CDR2 shown in SEQ ID NO:5, and the VH CDR3 shown in SEQ ID NO:6; With

B. containing the VL CDR1 shown in SEQ ID NO:7, the VL CDR2 shown in SEQ ID NO:8; With the VL CDR3 shown in SEQ ID NO:9,

Wherein said antibody or its Fab can specifically in conjunction with people IL-6 (e.g., can specifically in conjunction with the site II of people IL-6).

3. the antibody of separation according to claim 1 or claim 2 or Fab, wherein said antibody or Fab can with the K of 240pM or lower _din conjunction with IL-6, and there is 70 DEG C or higher T _m.

4. antibody according to claim 1 or claim 2 or Fab, wherein said antibody or Fab can with the K of 240pM or lower _din conjunction with IL-6, and there is 80 DEG C or higher T _m.

5. claim 1, claim 2, claim 3 or antibody according to claim 4 or Fab, at least one of R24, K27, Y31, D34, S118 or V121 of wherein said antibody or its Fab and people IL-6 combines.

6. claim 1, claim 2, claim 3 or antibody according to claim 4 or Fab, wherein said antibody or its Fab are in conjunction with R24, K27, Y31, D34, S118 and V121 of people IL-6.

7. the IL-6 antibody be separated or its Fab, its

A. according to the mensuration that surperficial plasmon resonates, can with the K of 240pM or lower _ddissociate with people IL-6, and

B. HEK-Blue in vitro ^tMcan with active with IL-6 in the IC50 of 255pM or lower during IL-6 measures.

8. the IL-6 antibody be separated or its Fab, it can contain the antibody of SEQ ID NO:1 and SEQ ID NO:2 or its Fab or the antibody containing SEQ ID NO:3 and SEQ ID NO:2. or its Fab to the combination of people IL-6 by competitive inhibition.

9. the antibody be separated or its fragment, it can specifically in conjunction with the site II of people IL-6, wherein said antibody or its fragment comprise: (a) comprises the light-chain variable domain of the aminoacid sequence identical with the aminoacid sequence at least 95% of SEQ ID NO:12, with the heavy chain variable domain comprising the aminoacid sequence identical with the aminoacid sequence at least 95% of SEQ ID NO:11.

10. the IL-6 antibody of the separation described in any one of claim 1-9 or its Fab, wherein said antibody or its Fab have the unit price Kd of 2nM or higher under pH5.5.

Antibody described in 11. any one of claim 1-10 or Fab, wherein said antibody is IgG2 antibody.

Antibody described in 12. any one of claim 1-11 or Fab, the FcRn that wherein said antibody or Fab have change compared with reference antibody or its fragment combines.

13. antibody according to claim 12 or Fabs, it is decline that wherein said FcRn combines compared with reference antibody.

Antibody described in 14. claims 12 or 13 or its Fab, the Fc structural domain of wherein said antibody or Fab is change compared with reference antibody.

15. antibody according to claim 1 or Fabs, wherein said antibody or its Fab are included in sudden changes one or more in H311, D313, I254 or H436 of SEQ ID NO:23.

16. antibody according to claim 15 or Fabs, wherein said antibody comprises the one or more sudden changes in the group being selected from and being made up of H311A, H311E, H311N and D313T, I254A, I254R and H436A.

17. 1 kinds of monoclonal antibodies be separated, it comprises SEQ ID NO:25.

18. 1 kinds of IL-6 antibody or its fragment be separated, it is specifically in conjunction with the site II of people IL-6, and the FcRn showing reduction compared with the corresponding antibody with wild-type Fc structural domain combines.

Antibody described in 19. aforementioned any one claims or Fab, be used for the treatment of the experimenter (as people) suffering from IL-6 relative disease.

20. antibody according to claim 19 or Fabs, wherein said disease is the eye disease that the IL-6 level to raise in vitreum is feature.

Antibody described in 21. claims 19 or 20 or Fab, be used for the treatment of and suffer from diabetic macular edema (DME), diabetic retinopathy, uveitis, dry eye syndrome, uveitis, age-related macular degeneration (AMD), proliferative diabetic retinopathy PDR (PDR), retinal vein occlusion (RVO), optic neuromyelitis (NMO), corneal graft, corneal abrasion or the physically impaired experimenter of eyes (as people).

22. antibody according to claim 21 or Fabs, are used for the treatment of the experimenter (as people) suffering from DME.

23. 1 kinds of compositions, it comprises antibody described in any one of claim 1-18 or Fab.

24. 1 kinds of compositions, it comprises antibody according to claim 1 or claim 2, and wherein said antibody is human monoclonal antibodies.

Composition described in 25. claims 23 or 24, it is used for the treatment of IL-6 relative disease.

26. compositions according to claim 25, it is used for the treatment of the eye disease that the IL-6 level to raise in vitreum is feature.

Composition described in 27. claims 23 or 24, it is used for the treatment of diabetic macular edema (DME), diabetic retinopathy, uveitis, dry eye syndrome, age-related macular degeneration (AMD), proliferative diabetic retinopathy PDR (PDR), retinal vein occlusion (RVO), optic neuromyelitis (NMO), corneal graft, corneal abrasion or eyes physical damnification.

28. 1 kinds of methods for the treatment of IL-6 relative disease, described method comprises IL-6 antibody described in from any one of claim 1-18 to experimenter's administering therapeutic significant quantity or its fragment.

29. methods according to claim 28, wherein said IL-6 relative disease is the eye disease that the IL-6 level to raise in vitreum is feature.

Method described in 30. claims 28 or 29, wherein said IL-6 relative disease is diabetic macular edema (DME), diabetic retinopathy, dry eye syndrome, age-related macular degeneration (AMD), proliferative diabetic retinopathy PDR (PDR), retinal vein occlusion (RVO), optic neuromyelitis (NMO), corneal graft, corneal abrasion or eyes physical damnification.

31. methods according to claim 29, are wherein delivered to the vitreum of subject eye by described antibody or Fab.

The method of 32. claims 29, wherein said IL-6 relative disease is diabetic macular edema and described antibody or its fragment is delivered to the vitreum of subject eye.

33. 1 kinds of carriers, it comprises the sequence of coding SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

34. 1 kinds of cells, it can express in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 one or more.

The method of the systematic effect of 35. 1 kinds of minimizing suppression IL-6 in experimenter, described method comprises to experimenter's administration of antibodies or its fragment, and described antibody or its fragment can suppress the activity of IL-6 and have the FcRn binding activities weakened with having compared with the corresponding antibody of wild-type Fc structural domain or its fragment.

The method of 36. claims 35, wherein K _d1 μM is greater than under the pH of 5 to 6.